Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of IFN-α

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1 JVI Accepted Manuscript Posted Online 9 August 2017 J. Virol. doi: /jvi Copyright 2017 American Society for Microbiology. All Rights Reserved. 1 Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of IFN-α Hisashi Akiyama 1, Nora-Guadalupe Pina Ramirez 1, Gregory Gibson 3, Christopher Kline 2, Simon Watkins 3, Zandrea Ambrose 2 and Suryaram Gummuluru Department of Microbiology, Boston University School of Medicine, Boston, MA Division of Infectious Diseases, Department of Medicine and 3 Center for Biologic Imaging and Department of Cell Biology and Molecular Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA *Corresponding author Suryaram Gummuluru, Ph.D. Department of Microbiology Boston University School of Medicine 72 E. Concord St., R512 Boston, MA Ph: (617) Fax: (617) rgummulu@bu.edu Running Title: CD169 and evasion from IFN-α-mediated inhibition of HIV Abstract: 250 Text: 9,311

2 Abstract A hallmark of HIV-1 infection in vivo is chronic immune activation concomitant with type I interferon (IFN) production. Although type I IFN induces an antiviral state in many cell types, HIV-1 can replicate in vivo via mechanisms that have remained unclear. We have recently identified a type I IFN-inducible protein CD169 as the HIV- 1 attachment factor on dendritic cells (DCs) that can mediate robust infection of CD4 + T cells in trans. Since CD169 expression on macrophages is also induced by type I IFN, we hypothesized that type I IFN-inducible CD169 could facilitate productive HIV-1 infection in myeloid cells in cis and CD4 + T cells in trans and thus offset antiviral effects of type I IFN. In support of this hypothesis, infection of HIV-1 or MLV-Env pseudotyped HIV-1 particles was enhanced in IFN-α-treated THP1 monocytoid cells, and this enhancement was primarily dependent on CD169- mediated enhancement at the virus entry step, an observation phenocopied in HIV-1 infections of IFN-α-treated primary monocyte-derived macrophages (MDMs). Furthermore, expression of CD169, a marker of type I IFN-induced immune activation in vivo, was enhanced in lymph nodes from RT-SHIV-infected pigtailed macaques, compared to uninfected macaques, and interestingly, there was extensive co-localization of p27 gag and CD169, suggesting productive infection of CD169 + myeloid cells in vivo. While cell-free HIV-1 infection of IFN-α-treated CD4 + T cells was robustly decreased, initiation of infection in trans via co-culture with CD169 + IFN-α-treated DCs restored infection suggesting that HIV-1 exploits CD169 in cis and in trans to attenuate a type I IFN-induced antiviral state. 2

3 Structured Abstract HIV-1 infection in human causes immune activation characterized by elevated levels of pro-inflammatory cytokines including type I interferons (IFN). Although type I IFN induces an antiviral state in many cell types in vitro, HIV-1 can replicate in vivo via mechanisms that have remained unclear. In this report, we test the hypothesis that CD169, a type I IFN-inducible HIV-1 attachment factor, offsets antiviral effects of type I IFN. Infection of HIV-1 was rescued in IFN-α-treated myeloid cells via upregulation of CD169 and subsequent increase in CD169-dependent virus entry. Furthermore, extensive co-localization of viral Gag and CD169 was observed in lymph nodes of infected pigtailed macaques, suggesting productive infection of CD169 + cells in vivo. Treatment of dendritic cell (DC) T cell co-cultures with IFN-α, upregulated CD169 expression on DCs and rescued HIV-1 infection of CD4 + T cells in trans suggesting that HIV-1 exploits CD169 to attenuate type I IFN-induced restrictions. 3

4 Introduction Innate immune responses play a crucial role in combating invading pathogens by sensing pathogen-associated molecular patterns, initiating signaling cascades culminating in secretion of numerous pro-inflammatory cytokines, that provide the first line of defense against infectious microbes. One of the key cytokines to combat viral pathogens is type I interferon (IFN) such as IFN-α. In the case of HIV-1 infection, IFN-α is secreted immediately after infection (1), mainly by plasmacytoid dendritic cells (pdcs) (2). pdcs are present both in circulation and at peripheral mucosal sites and upon sensing pathogens such as HIV-1, they secrete robust amounts of IFN-α ((3, 4) and reviewed in (5)). In addition to the acute phase of infection, blood IFN-α level is also elevated in the chronic phase of HIV-1 infection (6, 7). In fact, a hallmark of HIV-1 infection is chronic immune activation characterized by elevated levels of various pro-inflammatory cytokines including type I IFNs (reviewed in (8)), and is correlated to a poor prognosis of HIV-1 disease progression (9). Many factors have been attributed to cause immune activation in vivo, including elevated levels of LPS/endotoxin that are translocated across a compromised gut epithelial barrier resulting in persistent activation of mononuclear phagocytes (10, 11), bacterial or viral co-infections (reviewed in (12, 13)), abortive infections of resting CD4 + T cells by HIV-1 (14), or chronic stimulation of plasmacytoid DCs resulting in continued production of IFN-α (15-17). The secretion of type I IFNs leads to expression of various genes called interferon stimulated genes (ISGs). More than 150 ISGs have been reported so far and many of them have been characterized to possess potent antiviral activities. For instance, Mx1 inhibits influenza virus infection (reviewed in (18)) and ISG15 blocks a number of enveloped virus infections (reviewed in (19, 20)). Several ISGs have been 4

5 reported to have anti-hiv-1 activities including APOBEC3G, Mx2/MxB, SAMHD1 and tetherin/bst-2 (21-28). Although HIV-1 has evolved to encode evasion mechanisms to overcome some of these restriction factors (26-28), ISGs including those that have yet to be identified can potently inhibit HIV-1 replication in vitro ((29) and others). In vivo, however, HIV-1 is capable of replicating even in the presence of type I IFNs, suggesting HIV-1 has exploited additional mechanisms to overcome the effects of ISGs. CD169/Siglec-1 is an ISG whose expression is induced by type I IFNs on myeloid cells such as macrophages and DCs (30, 31). Recently, work by us and others have identified CD169 as the type I IFN-inducible HIV-1 attachment factor on myeloid dendritic cells (DCs) that captures virus particles and targets them to the DC-mediated trans-infection pathway (31, 32). CD169 specifically recognizes terminal 2,3-linked sialic acid residues such as those present in the glycosphingolipid GM3 (30), the HIV-1 particle-associated ligand necessary for virus capture by CD169 (33, 34). The CD169 GM3 interaction leads to accumulation of HIV-1 virions in a non-lysosomal compartment and mediates a robust infection of T cells when HIV-laden-mature DCs (mdcs) are co-cultured with target CD4 + T cells, a mechanism of trans-infection (31, 32). While CD169 is constitutively expressed by certain tissue resident macrophages including sub-capsular sinus macrophages in lymphoid tissues (reviewed in (35)), it is well-appreciated that CD169 expression levels are correlated with type I IFN levels and inflammatory disease progression in vivo (36, 37). In HIV-1 infection in humans and experimental infection of macaques with SIV, induction of CD169 expression in peripheral blood monocytes has been observed in early stages of infection and the expression has remained high only in the case of pathogenic lentiviral infections (38, 39). Furthermore, recent studies have 5

6 demonstrated a critical role for CD169 + cells in retroviral spread in vivo (40). Thus, it has been postulated that CD169 is not only a biomarker of pathogenic lentiviral infections, but might also contribute to HIV-1 pathogenesis in vivo. In this study, we hypothesized that type I IFN-inducible CD169 can attenuate the antiviral effects of type I IFNs by enhancing HIV-1 infection in myeloid cells in cis and in trans. To test this hypothesis, we used a monocytic cell line, THP-1, as a model system and showed that treatment of THP-1 cells with IFN-α induced expression of CD169, and surprisingly, enhanced wild type HIV-1 replication even in the presence of IFN-α by facilitating enhanced fusion and entry of virus particles. Interestingly, enhanced entry of HIV-1 in the presence of IFN-α was dependent on the route of infection because VSV-G pseudotyped HIV-1 lentivector was severely attenuated for fusion and replication in IFN-α-treated THP-1 cells. Furthermore, evasion from an IFN-α-induced anti-viral state in primary monocyte-derived macrophages (MDMs) was also partially dependent on CD169-mediated enhancement of HIV-1 particle fusion in MDMs. Finally, induced expression of CD169 on inflammatory DCs enhanced virus access to the DC-mediated transinfection pathway and facilitated virus replication in CD4 + T cells, even in the presence of type I IFN. These findings suggest an important role of type I IFNinducible CD169 expression on myeloid cells in attenuating the antiviral effects of type I IFNs on HIV-1 replication. 6

7 Materials and Methods Ethics Statement This research has been determined to be exempt by the Institutional Review Board of the Boston University Medical Center as it does not meet the definition of human subjects research, since all human samples were collected in an anonymous fashion and no identifiable private information was collected. Experimental procedures on 13 pigtailed macaques (Macaca nemestrina) used in the study were performed at the National Institutes of Health or the Washington National Primate Center, in both previous studies (41, 42) and a new study, with approval by the Institutional Animal Care and Use Committee at each institution (IACUC approval # ). The animals were negative for serum antibodies to HIV type 2, simian immunodeficiency virus (SIV), type D retrovirus, and simian T-lymphotropic virus type 1 at study initiation and were cared for in accordance with the American Association of Accreditation of Laboratory Animal Care standards. Briefly, all animals were housed in an AAALAC accredited facility and in compliance with the guidelines in the Guide for the Care and Use of Laboratory Animals. Animals were maintained in Animal Biosafety Level 2 housing according to the provisions of the 5 th edition of the Biosafety in Microbiological and Biomedical Laboratories. Filtered drinking water was available ad libitum, and a standard commercially formulated nonhuman primate diet was provided thrice daily and supplemented 3-5 times weekly with fresh fruit and/or forage material as part of the environmental enrichment program. Each cage contained a perch, a two portable enrichment toys, one hanging toy, and a rotation of additional items (including stainless steel rattles, mirrors, and challenger balls). Additionally the animals were able to listen to radios during the light phase of their day. Pain and distress were relieved by appropriate measures. Animals that were 7

8 diagnosed by the veterinarian to be experiencing more than momentary pain and distress were evaluated and treated with appropriate analgesic drugs as indicated. The end point of the study was euthanasia at the stated time point by intravenous pentobarbital (80 mg/kg) in the saphenous vein after sedation with 3 mg/kg Telazol Plasmids HIV-1 Lai-luc (a CXCR4-tropic infectious proviral construct encoding luciferase in place of nef orf), HIV-1 Lai env-luc (Env deficient HIV-1 Lai containing a luciferase reporter gene in place of the nef orf), HIV-1 NL4-3 and HIV-1 Lai (CXCR4-tropic viruses), Lai/Balenv (CCR5-tropic), Lai/Balenv-luc, HIV-1 Lai/YU-2env (CCR5-tropic) and psiv3 + (SIVmac239 Vpx encoding SIV packaging vector) have been described previously (43-45). HIV-1 Lai/YU-2env-luc (HIV-1 Lai/YU-2env encoding luciferase in place of nef orf) was created by replacing SalI-BamHI fragment of HIV-1 Lai-luc with corresponding fragment of HIV-1 Lai/YU-2env. The CCR5-tropic HIV gp160 (Bal Env) expression vector, the CXCR4-tropic HIV gp160 (Lai Env) expression vector, the ampho-tropic MLV (MLV-A) Env expression vector and the VSV-G expression vector were described previously (45, 46). plnc/cd169 (a retrovirus vector expressing human CD169) was described previously (31). plnc/ccr5, a retrovirus vector expressing human CCR5, was constructed as follows. Human CCR5 cdna was PCR amplified using the following primer set: CCR5-HindIII-F, 5'-TTTAAGCTTG CCACCATGGA TTATCAAGTG TCAAGTCC-3' and CCR5-NotI-R, 5'- TTGCGGCCGC TCACAAGCCC ACAGATATTT CC-3'. The PCR fragment was digested with HindIII and NotI and cloned into a retrovirus expression vector plncx digested with the same restriction endonucleases. 8

9 Cells Human inflammatory dendritic cells (IFN-DCs) were derived from CD14 + peripheral blood monocytes by culturing in the presence of GM-CSF (1400 U/ml; Genzyme) and IFN-α (1000 U/ml; PBL Interferon Source) for 3 days, as described previously (31). Human MDMs were derived from CD14 + peripheral blood monocytes by culturing in RPMI1640 (Invitrogen) containing 10% heat-inactivated human AB serum (Gemini Bio Products) and recombinant human M-CSF (20 ng/ml; Peprotech) for 5 days. Primary human CD4 + T cells were positively isolated from CD14-depleted PBMCs using CD4-conjugated magnetic beads and LS MACS cell separation columns (Miltenyi Biotech), activated with 2% PHA (Invitrogen) for 2 days, washed and cultured in RPMI containing 10% FBS and human ril-2 (50 U/ml, NIH AIDS Research and Reference Reagent Program). HEK293T, U87/CD4/CCR5 (NIH AIDS Research and Reference Reagent Program), THP-1 (human monocytic cell line, clone ATCC, obtained from the NIH AIDS Research and Reference Reagent Program) have been described previously (31, 47). To create a THP-1 cell line constitutively expressing CCR5 (THP-1CCR5), THP-1 cells were transduced by VSV- G pseudotyped plnc/ccr5 retrovirus vector (see above), cultured in the presence of G418 (1 mg/ml, HyClone), and cells expressing high levels of CCR5 were FACS sorted using a FACS AriaIII (BD) as previously described (31). To create a U87/CD4/CCR5 cell line expressing CD169, U87/CD4/CCR5 cells were transduced with VSV-G pseudotyped plnc/cd169 retrovirus vector, cultured in G418 (1 mg/ml) containing media and further selected for high CD169 expression by anti-cd169 conjugated magnetic beads and MACS (Miltenyi), as previously described (34). For measuring surface CD169 expression levels, cells were stained using Alexa488 9

10 conjugated anti-cd169 (AbD Serotec) and analyzed with FACS Calibur (BD) or Alexa647 conjugated anti-cd169 (BioLegend) and analyzed with LSRII (BD) Viruses Replication competent viruses, HIV-1 NL4-3, HIV-1 Lai, HIV-1 Lai-luc, HIV-1 Lai/YU- 2env, HIV-1 Lai/YU-2env-luc, HIV-1 Lai/Balenv, HIV-1 Lai/Balenv-luc, and SIV mac239 Vpx containing virus like particles (VLPs) were derived via calcium phosphatemediated transient transfection of HEK293T cells, as described previously (34). HIV- 1 vectors pseudotyped with HIV-1 Env, MLV-A Env and VSV-G were generated from HEK293T cells via co-transfection of HIV-1 Lai env-luc with HIV-1 Lai Env, HIV-1 Bal Env, MLV-A Env, or VSV-G expression plasmid, respectively. For the virus fusion assay described below, HEK293T cells were co-transfected with HIV-1 infectious constructs with pmm310, a plasmid encoding BlaM-Vpr fusion protein (48), or HIV-1 Lai env-luc, Env-expression construct and pmm310. Virus-containing cell supernatants were harvested 2 days post-transfection, cleared of cell debris by centrifugation (300 x g, 5 min), passed through 0.45 µm filters, and stored at -80 C until further use. For some experiments, viruses in the supernatants were concentrated by ultracentrifugation on a 20% sucrose cushion [24,000 rpm and 4 C for 2 hours with a SW28 rotor (Beckman Coulter)]. The virus pellets were resuspended in PBS, aliquoted and stored at -80 C until use. The capsid content of HIV-1 was determined by a p24 gag ELISA (45). Infection THP-1 cells (typically 2x10 5 cells) were infected with ng p24 gag of virus for 2 hours at 37 C, washed with PBS twice and cultured for 2 days before cells were 10

11 lysed and cell lysates used for measurement of luciferase activity. MDMs were seeded at 5-7x10 4 cells/well in a 96 well plate on the day before infection and left untreated or infected with SIVmac239 VLPs for 2 hours, washed, treated with antibodies (see below), infected with HIV-1 Lai/Balenv-luc or HIV-1 Lai/YU-2env-luc (5 ng/1x10 4 cells of p24 gag ) for 2 hours, washed and cultured for 3 days. To investigate DC-mediated trans-infection of T cells, inflammatory DCs (1x10 5 ; see above) were incubated with virus (10-20 ng p24 gag ) for 2 hours at 37 C in RPMI/10% FBS, washed 4 times with PBS and co-cultured with autologous CD4 + T cells at a 1:1 cell ratio in RPMI/10% FBS with ril-2. Luciferase activity in the cell lysates was measured using Bright-Glo (Promega). The assays were performed with a minimum of three independent donors. To competitively inhibit HIV-1 CD169 interaction, cells were pre-incubated with anti-cd169 (7D2, Novus Biologicals) monoclonal antibody (10 µg/ml) for 30 minutes at room temperature prior to virus exposure. Virus fusion assay To investigate HIV fusion to target cells, a FACS based assay was utilized, as previously described (48). Briefly, HIV-1 particles containing BlaM-Vpr fusion protein was used to infect target cells. After incubating 2-4 hours at 37 C, cells were washed with CO2-independent media (Invitrogen) and CCF2-containing media was added, incubated at 18 C over night, washed, fixed with 4% PFA and analyzed by LSRII (BD). For DC-T transfer assay, the cells were stained for CD11c (BD) and CD3 (BD) and virus fusion in target CD4 + T cells (CD11c - CD3 + ) was analyzed. Immunoblot Analysis. 11

12 To assess expression of Mx2, cell lysates containing 30 µg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with a goat anti-mx2 antibody (Santa Cruz) followed by donkey anti-goat-igg-irdye 800CW (Li-Cor). As loading controls, actin was probed using rabbit anti-actin (SIGMA) followed by a goat anti-rabbit-igg-irdye 800CW (Pierce). The membranes were scanned with an Odessy scanner (Li-Cor). RT-SHIV infection of macaques The derivation of the RT-SHIVmne027 stock was previously described (41). Five of 13 macaques were infected intravenously with 1 x 10 5 infectious units as determined on TZM-bl cells and remained viremic throughout the study and at the time of necropsy, which was performed 26 to 49 weeks post-infection. Peripheral blood and multiple mesenteric lymph nodes (LNs) were removed from each animal at the time of necropsy. Plasma was separated by centrifugation and stored at -80 C until RNA extraction, while tissues were flash frozen dry in liquid nitrogen until RNA extraction or sectioning. RNA was extracted as previously described from plasma (49, 50) and tissues (42). Immunofluorescence tissue staining and quantification Mesenteric LNs were thawed in 2% paraformaldehyde for 2 hours, followed by overnight incubation in 30% sucrose at 4 C and shock frozen in liquid nitrogen cooled 2-methylbutane prior to sectioning at 10 μm thickness. Sections were blocked for 1h in 2% bovine serum albumin and 0.1% Triton X-100 and stained for 1h with anti-cd169 antibody (1:500; 7D2, Novus Biologicals), followed by 1h in Alexa488- condjugated goat anti-mouse IgG (1:500; A11029, Molecular Probes). Nonspecific 12

13 antibody binding was blocked by overnight incubation at 4 C with goat anti-mouse IgG Fab fragment ( , Jackson Immunoresearch) and then stained for 1h anti-siv Gag (KK64; NIH AIDS Reagent Program) (51), followed by 1h in Cy3- condjugated goat anti-mouse IgG (1:1000; , Jackson Immunoresearch). Finally, sections were stained for 1 min with DAPI and coverslipped with Gelvatol. Large image widefield scans (5 fields x 5 fields) were taken for each tissue with a 40X objective on a Nikon Eclipse Ti microscope with a Hamamatsu HQ2 camera. Nikon NIS-Elements software was used to count cells as previously described (52). Intracellular viral and IP-10 quantitation Viral RNA was quantified by quantitative RT-PCR (qrt-pcr) using RT-SHIV gag-specific primers as previously described for plasma (49, 50) and tissues (42). IP-10 was quantified by custom digital molecular barcoding using the NanoString ncounter System (NanoString Technologies Inc., Seattle, WA), as previously described (53). Briefly, a custom CodeSet was designed against macaque inflammatory and immune marker genes and "housekeeping" genes. Each assay also contained External RNA Controls Consortium transcript sequences as positive and negative hybridization controls. Samples were normalized to 100ng of input RNA in 5μl and were hybridized with reporter and capture probes at 65 C for 16h and then transferred to the NanoString ncounter Prep Station for removal of excess probes and non-target RNA, immobilization of the sample on the assay cartridge, and electrophoresis to linearize hybrids for barcode counting. Cartridges were placed onto the NanoString ncounter Digital Analyzer where images of the immobilized fluorescent reporters are collected by a CCD camera focused through a microscope objective lens. Images were collected for 600 fields of view per sample and 13

14 processed for data export. Normalization and data analysis was carried out with the NanoString ncounter Analysis Software v Quantification of viral RT products and 2LTR For the quantification of viral DNA synthesis and 2LTR circle formation (a marker for nuclear import of viral DNA), THP-1 cells (1x10 6 cells) were infected with 1000 ng p24 gag equivalents of virus for 2 hours at 37 C, washed with PBS twice and cultured for 24 hours before cells were lysed for DNA extraction with a DNeasy kit (QIAGEN). As a background control, THP-1 cells were treated with 5 µm AZT (NIH AIDS Research and Reference Reagent Program) for at least 15 min prior to infection. Late RT products were amplified using the following primer sets; MH531_Late_F: 5'- TGTGTGCCCG TCTGTTGTGT-3', MH532_Late_R: 5'- GAGTCCTGCGTCGAGAGAGC-3', and a probe, LRT_P: 5 -(FAM)- CAGTGGCGCCCGAACAGGGA-(TAMRA)-3 as previously described (54). 2LTR circles were amplified using the following primer sets and a probe as reported; 2LTR_F_MH535: 5'-AACTAGGGAA CCCACTGCTTAAG-3', 2LTR_R_MH536: 5'- TCCACAGATC AAGGATATCTTGTC-3', 2-LTR probe_mh603: 5 -(FAM)- ACACTACTTGAAGCACTCAAGGCAAGCTTT-(TAMRA)-3. As standards, serial dilution of equivalent plasmids, phiv-1 Lai and pltr-bru (kindly provided by Dr. Masahiro Yamashita, ADARC) for late RT products and 2LTR circles, respectively, were used. The amplicon copy number was normalized to input DNA amount. 14

15 Results IFN-α treatment of THP-1 cells enhanced HIV-1 infection. Cells exposed to IFN-α upregulate a number of antiviral ISGs, including IFITM1 and - 3 (can block virus entry, reviewed in (55)), members of the APOBEC family (can block reverse transcription, reviewed in (56)), and Mx2/MxB (can block nuclear import of viral pre-integration complex (21, 23, 25)) that restrict HIV-1 at early steps in the virus life cycle. In contrast, CD169/Siglec1 is an ISG, whose expression is induced on myeloid DCs upon IFN-α treatment (31) and has been previously shown to enhance HIV-1 replication by targeting captured virus particles to the DCmediated trans-infection pathway (31, 32). To investigate if CD169 expression could offset the effect of antiviral ISGs, we cultured monocytoid cell lines, THP-1 and THP- 1CCR5, in the presence of IFN-α for 48 h prior to virus exposure. Note that expression of CD169 and antiviral ISG, Mx2/MxB, was robustly induced upon IFN treatment (Figure 1A and B, respectively). To determine the extent of HIV-1 infection in IFN-αtreated THP-1 cells, cells were infected with HIV-1 carrying VSV-G, CCR5 (R5)- tropic HIV-1 Env (Bal or YU-2), CXCR4 (X4)-tropic HIV-1 Env (Lai) or ampho-tropic MLV (MLV-A) Env. As previously reported (23, 29), infection with VSV-G pseudotyped HIV-1 was severely attenuated in IFN-α-treated THP-1 cells (THP- 1/IFN) and the suppression (>50-fold) was observed even at high MOIs (Figure 1C). In contrast, infection with R5-tropic HIV-1 Lai/YU-2env was not reduced by IFN-α treatment (Figure 1D and F) and infection of another R5-tropic HIV-1 (Bal) was only mildly attenuated (~30% decrease) in IFN-α-treated THP-1CCR5 cells (THP-1CCR5/IFN) (Figure 1F). Interestingly, HIV-1 pseudotyped with HIV-1 Lai Env (X4) infection was even enhanced in THP-1/IFN cells compared to untreated THP-1 cells and the enhancement (~2-3 fold) was observed at all virus inputs (Figure 1E and G). 15

16 Furthermore, infection of HIV-1 pseudotyped with MLV-A Env was also enhanced in THP-1/IFN cells (Figure 1G). Also, staining for intracellular p24 gag in infections with two replication competent X4-tropic viruses, HIV-1 Lai and HIV-1 NL4-3 revealed IFN-α-dependent enhancement of virus infection in THP-1 cells (Figure 1H) Fusion of HIV-1 was enhanced in IFN-α-treated THP-1. To determine the step of HIV-1 replication cycle in THP-1 cells that was differentially affected upon IFN-α treatment, we quantified level of virus binding, virus entry, reverse transcription and nuclear import in cells infected with VSV-G-pseudotyped HIV-1 or HIV-1 Lai (X4) or HIV-1 Lai/Balenv (R5). While the amount of late RT products in VSV-G pseudotyped HIV-1 infection was significantly reduced in THP- 1/IFN cells compared to untreated cells (Figure 2A), there was no reduction (X4- tropic HIV-1 Lai infection) or only a slight reduction (R5-tropic HIV-1 Lai/Balenv infection) in late RT products in THP-1/IFN cells or THP-1CCR5/IFN cells (Figure 2A), which is consistent with the infection results (Figure 1). Furthermore, there was an additional decrease in 2LTR circle formation in VSV-G pseudotyped HIV-1 infections of THP-1/IFN cells compared to untreated cells (Figure 2B), suggesting nuclear import of viral DNA was inhibited by pre-treatment with IFN-α. However, there was no further enhancement or reduction in 2LTR circle formation in HIV-1 Lai infection (X4) in THP-1/IFN cells and in HIV-1 Lai/Balenv infection in THP-1CCR5/IFN cells (Figure 2B), suggesting IFN-α treatment did not affect nuclear import of viral DNA of R5- or X4-tropic HIV-1 in THP-1 cells. Interestingly, IFN-α-treatment of THP-1 cells resulted in enhanced virus particle binding irrespective of the pseudotyping Env (Figure 2C), consistent with our previous findings that IFN-α-induced expression of CD169 results in enhanced Env- 16

17 independent, GM3-dependent virus capture by DCs (31). Next we investigated the effect of IFN-α treatment on HIV-1 fusion in THP-1 cells. THP-1, THP-1/IFN, THP- 1CCR5 or THP-1CCR5/IFN cells were infected with BlaM-Vpr containing HIV-1 particles carrying VSV-G, MLV-A Env, R5-tropic HIV-1 Env (Bal or YU-2) or X4-tropic HIV-1 Env (Lai or NL4-3), and the extent of virus particle fusion was quantified by a FACS assay (48). While fusion of VSV-G pseudotyped HIV-1 in THP-1/IFN or THP- 1CCR5/IFN cells was significantly reduced (Figure 2D and E), we observed a slight enhancement in fusion of HIV-1 particles pseudotyped with Bal Env in THP- 1CCR5/IFN cells (Figure 2D) and a more substantial enhancement in fusion of Lai Env (> 5-fold) or MLV-A Env (> 10-fold) pseudotyped virus particles in THP-1/IFN cells (Figure 2E). Furthermore, entry of replication competent HIV-1 carrying R5 Env (Lai/YU-2env) or X4 Env (Lai and NL4-3) was also enhanced in THP-1CCR5/IFN or THP-1/IFN cells, respectively (Figure 2F). These results suggest that enhanced binding and fusion of HIV-1 carrying R5-tropic Env (Bal and YU-2), X4-tropic Env (Lai and NL4-3) or MLV-A Env in IFN-α-treated THP-1 cells can offset effects of ISGs and that the route of cellular entry engaged by virus particles is a critical determinant of HIV-1 evasion of early IFN-α inducible restrictions in THP-1 cells. Enhancement of virus entry and replication in IFN-α-treated THP-1 cells is mediated by CD169. CD169 expression is induced by type I IFN on primary myeloid cells and THP-1 cells (Figure 1A) and enhances HIV-1 capture by those cells (Figure 2C and (31, 32, 57, 58)). To determine if induced expression of CD169 on THP-1/IFN cells mediated the enhancement of HIV-1 fusion and replication, we pretreated THP-1/IFN cells with anti-cd169 blocking antibody (31), prior to virus exposure. As described above, IFN- 17

18 α pre-treatment decreased virus fusion and entry of VSV-G pseudotyped HIV-1 in THP-1/IFN cells, and this reduction was not affected by blocking CD169 (Figure 3A). In contrast, enhanced entry and fusion of HIV-1 carrying R5-tropic Envs, Bal (Figure 3B) or YU-2 (Figure 3C), X4-tropic Env (Lai; Figure 3D) or MLV-A Env (Figure 3E) was completely abrogated by pre-treatment of the cells with anti-cd169 blocking antibody. It is interesting to note that R5-tropic HIV-1 entry was dependent on basal expression of CD169 on untreated THP-1CCR5 (Figure 1A) since anti-cd169 blocking antibody treatment reduced entry to untreated THP-1 cells (Figure 3 B and C). In concordance with the effects of CD169 on virus entry and fusion, pretreatment with anti-cd169 antibody did not affect infection of THP-1/IFN cells by VSV-G pseudotyped HIV-1 (Figure 3F). In contrast, anti-cd169 blocking antibody reduced infections of both HIV-1 with Bal Env and YU-2 Env by 70% in untreated THP-1 cells (Figure 3G and H). Furthermore, anti-cd169 antibody reduced HIV-1 Lai/YU-2env infection by > 95% in THP-1/IFN cells (Figure 3H). These results suggest that basal and IFN-induced expression of CD169 enhances infection of R5- tropic HIV-1 and reduce (Bal Env) or offset (YU-2 Env) the antiviral effects of type I IFN in THP-1 cells. Interestingly, IFN-α-induced expression of CD169 greatly enhanced infection of both X4-tropic HIV-1 Lai Env (Figure 3I) and MLV-A Env (Figure 3J) pseudotyped HIV-1 in THP-1/IFN cells and resulted in approximately 4- fold higher infection than that in THP-1 cells. HIV-1 particles produced from cells treated with PDMP, a competitive inhibitor of GSL synthesis (45) have reduced levels of virion-associated GSLs and are attenuated for capture by CD169 (31, 33, 34). When THP-1/IFN cells were infected with GSL-deficient HIV-1 (PDMP), IFN-αdependent enhancement of virus infection in THP-1 cells was abrogated (Figure 3K). 18

19 These results suggest the CD169 GSL interaction dependent enhancement of virus entry rescues HIV-1 replication in THP-1/IFN cells Exogenous expression of CD169 attenuated the antiviral effects of IFN-α. To further investigate the role of CD169 in attenuating the anti-hiv-1 effects of ISGs, U87 cells expressing CD4 and CCR5 were retrovirally transduced to constitutively express CD169 (U87/CD169; Figure 4A). We chose this cell line because previous studies have demonstrated suppression of HIV-1 infection in U87 cells upon pretreatment with IFN-α (21). Pre-treatment of U87 or U87/CD169 cells with IFN-α robustly induced Mx2/MxB expression (Figure 4B). While IFN-α pre-treatment inhibited HIV-1 infection in U87 cells by about 70% (Figure 4C), exogenous expression of CD169 in U87 cells (U87/CD169) enhanced HIV-1 infection even in the presence of IFN-α (Figure 4C), suggesting CD169-specific interaction with HIV-1 can attenuate the IFN-α-induced antiviral state in target cells. Type I IFN-inducible expression of CD169 on primary MDMs partially offsets antiviral effects of IFN-α. In order to investigate the role of CD169 in attenuating antiviral effects of ISGs in primary cells, MDMs were treated with increasing doses of IFN-α for 2 days prior to virus challenge. Expression of CD169 on MDMs was induced in a dose-dependent manner upon IFN-α treatment (Figure 5A). We next examined if induced expression of CD169 on MDMs enhanced HIV-1 fusion. MDMs were treated with 100 U/ml IFNα prior to challenge with BlaM-Vpr containing replication competent R5-tropic HIV-1 (Bal or YU-2). IFN-α pretreatment of MDMs enhanced HIV-1 fusion in MDMs (YU-2 Env, Figure 5D), in agreement with previous findings (29), while minimally impacting 19

20 Bal Env-mediated fusion (Figure 5C). Interestingly, initiation of virus infection of IFNα-treated MDMs in the presence of anti-cd169 blocking antibody reduced HIV-1 Lai/Balenv and HIV-1 Lai/YU-2env fusion by approximately 1.7-fold and 2-fold, respectively (Figure 5B, C and D). To investigate the role of CD169 in virus infection of IFN-α-exposed MDMs, MDMs were pretreated with increasing doses of IFN-α and infected with HIV-1 Lai/Balenv-luc (Figure 5E) or HIV-1 Lai/YU-2env-luc (Figure 5F) reporter virus in the presence of anti-cd169 blocking antibody. HIV-1 Lai/Balenv replication was dependent of basal expression of CD169 on IFN-untreated MDMs since CD169 antibody reduced its infection by 30% (Figure 5E), while there was no significant effect on Lai/YU-2env infection (Figure 5F). Although IFN-α potently reduced HIV-1 infection in MDMs particularly in the absence of CD169 (α-cd169), the presence of CD169 (IgG) restored virus infection (about 2-fold), particularly that of Lai/YU-2env from 27% (α CD169) to 45% (IgG) of that of untreated cells (10 U/ml IFN-α; Figure 5F). SAMHD1, whose expression can be induced by type I IFN in a cell-type dependent manner (59, 60) can potently restrict replication of HIV-1 in macrophages (22, 24) and, therefore, may mask the effect of other ISGs on viral replication. Hence, to examine the effect of CD169 on antiviral ISGs in the absence of SAMHD1, IFN-α-treated MDMs were exposed to SIVmac239 Vpx-containing VLPs to degrade SAMHD1 (22, 24) prior to infection with HIV-1. When HIV-1 interaction with CD169 was blocked with anti-cd169 blocking antibody, virus replication in IFN-αtreated MDMs was reduced to about 10% (both Lai/Balenv and Lai/YU-2env) of that observed with infection of untreated MDMs (100 U/ml IFN-α; Figure 5E and F). In contrast, preserving HIV-1 interactions with CD169 (IgG) reduced HIV-1 replication in MDMs treated with high dose of IFN-α only to 20% (Lai/Balenv) and only to 33% (Lai/YU-2env) of that observed in untreated MDMs (Figure 5E and F). Furthermore, 20

21 CD169-dependent rescue of virus replication was observed at all IFN-α inputs (Figure 5E and F). These results suggest that enhanced CD169 expression in IFNα-exposed MDMs increases virus particle fusion and partially alleviates IFN-αinduced blocks to HIV-1 replication in MDMs CD169 + cells in lymph nodes were infected with SHIV and display type I IFN signatures in vivo. To investigate the role of CD169 + cells in HIV-1 replication in vivo, we used SHIV infection of macaques as a model of HIV-1 infection in humans. Pigtailed macaques were infected with RT-SHIVmne027, a macrophage tropic SIV carrying HIV-1 RT (41). All infected monkeys displayed high plasma viremia (2.5 x10 5 ± 1.8 x10 5 copies/ml, means ± SEM, n=5) and high ongoing replication in lymph nodes (6.0 x10 6 ± 3.1 x10 6 copies/200 ng RNA, means ± SEM, n=5) at the time of necropsy (Table 1). Importantly, infected macaques showed a significantly higher IP-10 expression in tissues such as mesenteric lymph nodes (LNs), which was indicative of type I IFN induction in tissues, than uninfected monkeys (Figure 6A). Figure 6B shows representative images of a mesenteric LN of uninfected monkeys and chronically SHIV-infected monkeys, which were stained for CD169 (green), p27 gag (red) and nucleus (DAPI; blue). Interestingly, number of CD169 + cells in the LN of infected monkeys was dramatically enhanced (6-fold) compared to LNs from uninfected macaques (Figure 6C). Intriguingly, about 30% of the p27 gag+ cells were also CD169 + (Figure 6D), suggesting that CD169 + cells might play an important role in establishment of SHIV replication in lymphoid tissues in vivo even in the presence of type I IFN responses. 21

22 CD169 cells on inflammatory dendritic cells attenuated anti-viral effects of ISGs by enhancing virus fusion to CD4 + T cell in trans. In addition to MDMs, DCs are known to up-regulate CD169 upon IFN-α stimulation (31, 43). In vivo, inflammatory conditions can drive monocyte differentiation into inflammatory DCs at local sites of infection (61-64). In vitro, inflammatory DCs can be differentiated from peripheral blood monocytes by culturing in the presence of IFN-α and GM-CSF (65, 66). As previously reported, CD169 on LPS-matured DCs and inflammatory DCs (referred as IFN-DCs in this study) can greatly enhance virus replication in CD4 + T cell in trans (31). Therefore, we sought to determine if CD169- dependent IFN-DC-mediated trans-infection could overcome the IFN-α-induced antiviral state in CD4 + T cells. Cell-free infection of IFN-treated CD4 + T cells by X4- tropic HIV-1 (Figure 7A) and R5-tropic HIV-1 (Figure 7B) was inhibited by 90% and 80%, respectively, compared to virus infection of untreated CD4 + T cells. In contrast, initiation of infection of IFN-α-treated CD4 + T cells in trans by IFN-DCs was only attenuated by 40-50% compared to infections of IFN-α-naive (untreated) CD4 + T cells (Figure 7A and B), suggesting that CD169-mediated DC-T cell transmission could attenuate the antiviral effects of ISGs in CD4 + T cells. Next, we examined the role of CD169 in the enhancement of HIV-1 replication in IFN-DC-mediated transinfection of IFN-α-treated CD4 + T cells. IFN-DCs were pretreated with anti-cd169 blocking antibody prior to virus exposure and initiation of co-cultures. Pretreatment of IFN-DCs with isotype-matched IgG control antibody did not affect HIV-1 replication in IFN-α-treated CD4 + T cells (Figure 7C). However, pretreatment of IFN-DCs with anti-cd169 blocking antibody greatly reduced HIV-1 replication in IFN-α-treated CD4 + T cells to levels less than that observed upon cell-free infection of IFN-αtreated CD4 + T cells (Figure 7C). 22

23 Finally, to investigate the mechanism of IFN-DC-mediated restoration of virus replication in IFN-α-treated CD4 + T cells, we measured HIV-1 fusion in CD4 + T cells upon initiation of infection in cis (cell-free infection) as well as in trans (IFN-DCmediated trans-infection). HIV-1 fusion was unaffected by IFN-α pre-treatment of CD4 + T cells, similar to previous studies (29), while IFN-DC-mediated HIV-1 fusion into CD4 + T cells was greatly enhanced (Figure 7D). Interestingly, anti-cd169 blocking antibody blocked IFN-DC-mediated HIV-1 fusion to IFN-α-treated CD4 + T cells, while isotype-matched control antibody did not affect virus fusion to IFN-αtreated CD4 + T cells in IFN-DC-mediated trans-infection (Figure 7D). These results suggest that CD169 on IFN-DCs mediated enhancement of HIV-1 fusion into IFN-αtreated CD4 + T cells, leading to a robust infection of IFN-α-treated CD4 + T cells and thus attenuating antiviral effects of ISGs. Discussion Although type I IFNs are generally thought to be beneficial against microbial infections, many recent studies have demonstrated detrimental effects of type I IFNs in persistent virus infections, including pathogenic lentiviral infections (44, 67-74). A hallmark of chronic HIV-1 infection is systemic immune activation, characterized by elevated levels of a number of pro-inflammatory cytokines including type I IFNs (75). In vitro, type I IFN can block HIV-1 infection and many of the ISGs have potent anti- HIV-1 activities (76). However, HIV-1 is still able to replicate even in the presence of IFN-α in vivo, though the mechanisms by which HIV-1 is able to persist have remained unclear. In this study, we demonstrate that in contrast to the previously defined restrictions to HIV-1 replication by ISGs, induction of CD169 expression partially ablated type I IFN-induced blocks to HIV-1 replication by enhancing HIV-1 23

24 fusion and entry within target cells. These findings highlight a unique role for CD169 amongst all of the ISGs in promoting virus replication. ISGs display potent anti-viral activities against diverse viruses. For example, Mx1/MxA prevents influenza virus infection (reviewed in (18)) and ISG15 inhibits a number of viruses including HIV-1 and Ebola virus (reviewed in (19, 20)). While infection of HIV-1 lentivectors pseudotyped with VSV-G was potently inhibited by IFN-α pre-treatment, HIV-1 Env- or MLV-A Env-pseudotyped lentivector infections were not affected or even enhanced in THP-1 cells. IFITM1, -2 and -3 are some of the well-characterized ISGs that restrict entry of a number of viruses including VSV and HIV-1 (reviewed in (55)). IFITM proteins are located in cellular membranes such as plasma membrane and endosomal membranes and susceptibility of virus to IFITM-mediated restriction depends on virus entry sites and IFITM localization. Amongst the five IFITM proteins found in humans, IFITM3 is localized in late endosomes/lysosomes and restricts VSV-G pseudotyped virus entry, but does not block MLV Env pseudotyped virus entry (77). Previous studies have reported that HIV-1 infection can be blocked by IFITM1, but the magnitude of its inhibitory effect was cell-type dependent (77-79). It is thus plausible that entry-site specific antiviral ISGs such as IFITM1 and IFITM3 are unable to restrict entry of viruses that fuse at the plasma membrane, such as HIV-1 or MLV, in THP-1 cells. Since VSV-G pseudotyped HIV-1 has been extensively used in studies for characterizing ISG functions against HIV-1, care should be taken in interpreting observed restrictions to virus infection, since effects of ISGs on VSV-G-dependent entry might overestimate the effects of ISGs on post-entry steps in HIV-1 replication. Majority of HIV-1 transmission occurs at mucosal sites after sexual intercourse (80). Recent studies have suggested that transmitter/founder (T/F) 24

25 viruses are relatively resistant to type I IFN (81, 82), though these findings have not been replicated in other studies (83-85). In this study, we found that there's a difference in CD169-mediated enhancement of entry in THP-1/IFN cells and rescue of infection in MDMs between two R5-tropic HIV-1 strains. It would be interesting to test T/F viruses for CD169-mediated enhancement in entry since it is possible that mucosal tissue-resident myeloid cells may express CD169 upon exposure to IFNinducing TLR-ligands such as LPS, which might aid in mucosal transmission of T/F viruses. Interestingly, X4-tropic HIV-1infection was also enhanced by type I IFN in THP-1 cells (Figure 1 to 3). HIV-1 infection is mostly initiated with R5-tropic strains and X4-tropic HIV-1 emerges towards the latter stages of infection in HIV-1 infected individuals (86, 87). It has been shown that X4-tropic strains may evolve preferentially in secondary lymphoid tissues such as lymph nodes in animal models (88, 89) where CD169 + macrophages preferentially reside (35). Moreover, the number of CD169 + monocytes increases in blood with disease progression (39). It is tempting to speculate that CD169-dependent enhancement of replication may play a role in X4-tropic HIV-1 evolution and spread at late stages of HIV-1 infection. Since, some X4-tropic HIV-1 isolates can infect MDMs (90, 91), it would be of interest to examine whether infection of X4-tropic clinical isolates is also enhanced by CD169- mediated trans-infection of CD4 + T cells or cis infections of CD169 + MDMs. CD169 binds to HIV particles via 2,3-linked sialic acid residues of glycosphingolipids, particularly GM3, on virus membranes (30, 33, 34). Although we have observed differential effects of IFN-induced CD169 on virus entry and infection in THP-1 cells and MDMs depending on Env, binding of HIV-1 carrying different Envs to THP-1 cells was similarly enhanced by CD169 (Figure 2), suggesting CD169 HIV-1 interaction was independent of Env glycoproteins which is consistent 25

26 with previous studies (31, 32). At present, the mechanisms to explain why the magnitude of enhancement in capture did not correlate to virus particle fusion/entry remain unclear. It is possible that expression levels of CCR5 and CXCR4 or kinetics of clustering of CCR5 and CXCR4 after HIV-1 CD4 engagement may define entry kinetics or entry efficiency of CD169-bound HIV-1 particles. Interestingly, we have observed differences between two R5-tropic Envs in virus entry and infection mediated by CD169 on THP-1 and MDMs. The amount of Env glycoproteins incorporated into virions was not significantly different between YU-2 and Bal Envs (data not shown). Although both Bal and YU-2 Envs utilize CCR5 as a co-receptor, it has been shown that there are differences in their CD4 dependency and sensitivity to fusion inhibitors (92), which might impact entry efficiency of CD169-bound R5- tropic HIV-1 in THP-1 cells and MDMs. Entry of HIV-1 was greatly enhanced by CD169 on THP-1 cells in cis (Figure 2 and 3). CD169 captures HIV-1 and CD169-bound HIV-1 is clustered in deep plasma membrane invaginations in mature DCs and THP-1 cells (46). Intimate CD169 HIV-1 association may reduce the dissociation rate of virus particles with host cell membranes and thus increase the efficiency of HIV-1 fusion into THP-1 cells at the plasma membrane or at sites deficient for IFITM expression. In addition to cis infection, HIV-1 entry into CD4 + T cells by DC-mediated trans-infection was also significantly enhanced. HIV-1 transmission between DCs and T cells occurs through a tight cell-to-cell junction called the virological synapse (93). Unique structures of the virological synapse (94) may concentrate HIV-1 particles in a limited area and prevent virus diffusion, which may accelerate and enhance fusion of HIV-1 into T cells. In fact, we found that not only the magnitude but also the kinetics of HIV- 1 fusion was enhanced in mature DC-mediated trans-infection compared to those in 26

27 cell-free infection. Whether site of virus entry and fusion in primary CD4 + T cells mediated upon DC-mediated trans-infection is distinct from that of cell-free virus (95) and results in virus access to intracellular trafficking mechanisms and nuclear import pathways that are resistant to Mx2/MxB restriction remains to be determined. Since IFN-I treated T cells are less susceptible, but not completely refractory to establishment of HIV-1 infection, CD169-mediated transfer of HIV-1 particles to T cells might enhance the effective MOI and thus increase the probability of infection of IFN-I treated T cells. In vivo, CD169 is constitutively expressed in macrophages particularly in tissue lymph nodes such as sub-capsular sinus (SCS) macrophages, perifollicular macrophages and medullary macrophages (reviewed in (35)). Furthermore, the frequency of CD169 + cells in blood is elevated in patients chronically infected with HIV-1 and, interestingly, in experimentally infected macaques with pathogenic strain of SIV but not with nonpathogenic strain (38, 39). Therefore it is plausible that CD169 + cells such as tissue resident macrophages play an important role in virus dissemination even in the presence of IFN-α in vivo. In line with this hypothesis, we found that about 30% of RT-SHIV-infected cells in peripheral lymph nodes were also CD169 + (Figure 6). It has long been appreciated that CD169 + SCS macrophages in the secondary lymph nodes are strategically positioned to intercept afferent lymphatics-borne pathogens (96-98). In support of his hypothesis, recent studies have demonstrated that murine CD169 + SCS macrophages retain enveloped virus particles including MLV, HIV-1 and VSV (vesicular stomatitis virus) on their cell surface following capture of lymph-borne virus after peripheral inoculation (40, 99, 100). Retention of intact undegraded antigens on the cell surface of CD169 + SCS macrophages and transfer to sub-adjacent naïve follicular B cells has been 27

28 hypothesized to be essential for generation of adaptive immune responses ( ). Furthermore, enforced replication of VSV in these cells is critical for host survival as it initiates localized production of IFNα/β (upon intracellular detection of VSV by pattern recognition receptors such as RIG-I (103)) and increases local viral antigen availability to promote innate and adaptive immune responses (104) before CD169 + SCS macrophages are killed by the cytopathic VSV. In contrast to the essential role of CD169 + SCS macrophages in initiating innate and adaptive immune responses to an acute virus infection (104), CD169 + cells in the draining lymphatic tissues and spleen can facilitate MLV trans-infection of B-1 cells in mice and HIV-1 infection of CD4 + T cells in humanized mice (40), suggesting retroviruses can parasitize CD169- dependent host defense mechanisms to facilitate systemic virus dissemination. While the molecular mechanisms by which lymph node SCS macrophages capture most pathogens remain unclear, CD169 expression is critical for capture of HIV-1 in a GM3 dependent manner (31-34). In contrast to the beneficial role of CD169 + myeloid cells in lytic virus infections such as VSV, capture and retention of HIV-1 by CD169 on the cell surface might facilitate cell-associated transfer of virus to CD4 + T cells. Alternatively, enhanced CD169-dependent HIV-1 fusion in CD169 + macrophages might result in establishment of tissue reservoir of productively infected macrophages that potentiate virus persistence. Thus, we hypothesize that HIV-1 capture and/or infection of CD169 + myeloid cells in the secondary lymph nodes and subsequent subversion of host protective responses might be a key mechanism for establishment of virus persistence. It would be of interest in future studies to test ability of CD169-specific neutralizing agents to reduce virus replication and persistence in vivo. 28

29 Acknowledgments We thank Dr. Masahiro Yamashita (Aaron Diamond AIDS Research Center) for the generous gift of the pltr-bru plasmid, Dr. Michael Emerman for MLV Env expression construct. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: AZT, THP-1ATCC from Drs. Li Wu and Vineet N. KewalRamani, HIV-1 immunoglobulin from NABI and National Heart Lung and Blood Institute (Dr. Luiz Barbosa), HIV-1 p24 gag hybridoma (183-H12-5C) from Dr. Bruce Chesebro, U87/CD4/CCR5 from Dr. HongKui Deng and Dr. Dan R. Littman, Human ril-2 from Dr. Maurice Gately, Hoffmann - La Roche Inc., pmm310 from Dr. Michael Miller and SIVmac251 Gag Monoclonal Antibody (KK64) from Dr. Karen Kent and Miss Caroline Powell. We thank the BUMC flow cytometry core facility for technical assistance. This work was supported by NIH grants AI (SG), AI (ZA) and GM (SCW). 29

30 Table 1. Summary of RT-SHIV infected monkeys. Monkey Plasma viral RNA (copies/ml) Viral RNA in mesenteric LNs (copies/200 ng RNA) M x x 10 6 A x x 10 7 A x x 10 6 T x x 10 6 GT x x 10 5 Means ± SEM 2.5 x10 5 ± 1.8 x x10 6 ± 3.1 x10 6 Downloaded from on August 16, 2018 by guest 30

31 Figure Legends Figure 1. IFN-α treatment of THP-1 cells enhanced HIV-1 infection. (A) Cell surface expression of CD169 on THP-1 and THP-1CCR5 cells untreated (NT) or treated with 1000 U/ml IFN-α (IFNα) was measured by flow cytometry. (B) Western blot analysis of THP-1 cell lysates untreated (NT) or treated with 1000 U/ml IFN-α (IFNα). Mx2 expression was quantified and normalized to the expression of actin, a loading control. Mx2/actin ratio of THP-1/IFN cells was further normalized to that of untreated THP-1 cells. Infection of HIV-1 pseudotyped with VSV-G (C), replication competent HIV-1 Lai/YU-2env-luc (D) or Lai-Env-pseudotyped HIV-1 in untreated (NT) or IFN-α-treated (IFNα THP-1 cells (C and E) or THP-1CCR5 cells (D). Cells were infected with increasing inputs of HIV-1 and replication was quantified 2 days post infection by measuring luciferase activity in cell lysates. The experiment was performed three times in duplicate and the data of a representative experiment is shown. (F) Infection of HIV-1 pseudotyped with VSV-G, HIV-1 Bal Env or replication-competent HIV-1 Lai/YU-2env-luc in untreated THP-1CCR5 (NT) and THP- 1CCR5/IFN (IFNα cells. The luciferase activity in THP-1 CCR5/IFN cell lysates 2 days post infection was normalized to that of THP-1CCR5 cells. The data shown are the means ± SEM of five (VSV-G), eight (HIV-1 Bal) or four (Lai/YU-2env) independent experiments performed in duplicate or triplicate. (G) Infection of HIV-1 pseudotyped with VSV-G, HIV-1 Lai Env or MLV-A Env in untreated THP-1 (NT) and THP-1/IFN 720 (IFNα cells. The luciferase activity in THP-1/IFN cell lysates 2 days post infection was normalized to that of THP-1 cells. The data shown are the means ± SEM of seven independent experiments performed in triplicate. (H) Infection of replicationcompetent HIV-1 Lai and HIV-1 NL4-3 in untreated THP-1 (NT) and THP-1/IFN (IFNα cells at 2 days post infection was quantified by intracellular p24 gag staining. 31

32 The data shown are the means ± SEM of four independent experiments. Two-tailed p values were calculated using one sample t-test (F and G) or paired t-test (H) in GraphPad Prism 5. *: p 0.05, **: p 0.01, ***: p 0.001, ns: not significant Figure 2. Exposure of THP-1 cells to IFN-α enhances HIV-1 fusion (A) Quantitative analysis of late RT products. Untreated (NT) or IFN-α-treated (IFNα THP-1 (for VSV-G or HIV-1 Lai) or THP-1CCR5 (for HIV-1 Bal) cells were infected with HIV-1 pseudotyped with VSV-G and replication competent HIV-1 Lai and HIV-1 Lai/Balenv and lysed at 24 h post infection for measurement of viral DNA by quantitative PCR. The amount of late RT products (A) or 2LTR circles (B) in IFN-αtreated THP-1 cells was normalized to that of untreated THP-1 cells. The data shown are the means ± SEM of three independent experiments. (C) Binding of HIV-1 particles pseudotyped with VSV-G (n=7, THP-1), HIV-1 Bal (n=5, THP-1CCR5), HIV-1 Lai (n=7, THP-1), MLV-A (n=7, THP-1) or replication competent HIV-1 Lai/YU-2env (n=3, THP-1CCR5) to untreated THP-1 (NT) or THP-1/IFN (IFNα cells was quantified by an ELISA and the percentage of binding was calculated. The data shown are the means ± SEM of independent experiments indicated above. (D) Virus fusion of HIV-1 pseudotyped with VSV-G or HIV-1 Bal Env in untreated THP-1CCR5 (NT) or THP- 1CCR5/IFN (IFNα cells was quantified. Each symbol represents the percentage of BlaM + cells obtained from an independent experiment and the means ± SEM are shown. (E) Virus fusion of HIV-1 pseudotyped with VSV-G, MLV-A Env or HIV-1 Lai Env in untreated THP-1 (NT) or THP-1/IFN (IFNα cells was quantified. Each symbol represents the percentage of BlaM + cells obtained from an independent experiment and the means ± SEM are shown. (F) Virus fusion of replication competent HIV-1 Lai/YU-2env (YU-2) in untreated THP-1CCR5 (NT) or THP-1CCR5/IFN (IFNα or HIV-1 32

33 Lai (Lai) and HIV-1 NL4-3 (NL43) in untreated THP-1 (NT) or THP-1/IFN (IFN cells was quantified. Each symbol represents the percentage of BlaM + cells obtained from an independent experiment and the means ± SEM are shown. Two-tailed p values were calculated using one sample t-test (A and B) or paired t-test (C-F) in GraphPad Prism 5. *: p 0.05, **: p 0.01, ***: p 0.001, ns: not significant. Figure 3. IFN-α-induced CD169 expression on THP-1 cells enhances HIV-1 fusion and infection. (A to E) Untreated (NT) THP-1 or THP-1CCR5 or IFN-treated (IFNα THP-1 or THP- 1CCR5 cells were incubated with isotype-matched control IgG (IgG) or anti-cd169 blocking antibody (αcd169) and infected with (A) VSV-G-pseudotyped HIV-1 (VSV- G), (B) Bal Env-pseudotyped HIV-1 (Bal), (C) replication competent HIV-1 Lai/YU- 2env (YU-2), (D) Lai Env-pseudotyped HIV-1 (Lai) or (E) MLV-A Env-pseudotyped HIV-1 (MLV-A) and virus fusion in these cells was quantified. Each symbol represents the percentage of BlaM + cells obtained from an independent experiment and the means ± SEM are shown. (F to J) Untreated (NT) THP-1 or THP-1CCR5 or IFN-treated (IFNα THP-1 or THP-1CCR5 cells were infected with (F) VSV-Gpseudotyped HIV-1 (VSV-G), (G) Bal Env-pseudotyped HIV-1 (Bal), (H) replication competent HIV-1 Lai/YU-2env (YU-2), (I) Lai Env-pseudotyped HIV-1 (Lai) or (J) MLV-A Env-pseudotyped HIV-1 (MLV-A) in the presence of either anti-cd169 blocking antibody (αcd169) or isotype-matched control IgG (IgG). Virus infection was quantified by measuring luciferase activity in cell lysates 2 days post infection and was normalized to the values in untreated cells (no IFNα data shown are the means ± SEM of independent experiments performed in. The 774 duplicate or triplicate (n=4 for F, n=5 for G, n=4 for H, n=6 for I, n=6 for J). (K) 33

34 Infection of HIV-1 pseudotyped with HIV-1 Lai Env produced from untreated cells (NT: GSL enriched virions) or PDMP-treated cells (PDMP: GSL deficient virions) in THP-1 (NT) and THP-1/IFN (IFNα. The luciferase activity in cell lysates 2 days post infection was normalized to that of untreated THP-1 cells. The data shown are the means ± SD of two independent experiments performed in triplicate. Twotailed p values were calculated using one-way ANOVA followed by the Tukey- Kramer post test (A-J, shown with lines) or the Dunnett's post test [F to J, shown just above each columns, comparing to untreated cells (first column)] in GraphPad Prism 5. *: p 0.05, **: p 0.01, ***: p 0.001, ns: not significant. Figure 4. Exogenous CD169 expression in U87 attenuates the effects of ISGs. (A) Cell surface expression of CD169 on parental U87 cells (U87) and CD169- transduced U87 cells (U87/CD169). (B) Western blot analysis of U87 cell or U87/CD169 cell lysates untreated (NT) or treated with 1000 U/ml IFN-α (IFNα). Mx2 expression was quantified and normalized to the expression of actin, a loading control. Mx2/actin ratio of IFN-α-treated U87 or U87/CD169 cells was further normalized to that of untreated cells. (C) Infection of HIV-1 pseudotyped with HIV-1 Bal Env in untreated U87, IFN-α-treated U87 or IFN-α-treated U87/CD169 cells was quantified 2 days post infection by measuring luciferase activity in cell lysates. The data shown are the means ± SD of two independent experiments performed in triplicate. Figure 5. Induced CD169 expression on MDMs enhances HIV-1 replication in the presence of IFN-α. (A) Cell surface expression of CD169 on MDMs treated with different doses of IFN-α. 34

35 (B) Representative FACS profiles of BlaM + MDMs pretreated with 100U/ml IFN-α for 2 days and infected with BlaM-Vpr containing HIV-1 Lai/YU-2env in the absence or presence of anti-cd169 blocking antibody (αcd169) or isotype-matched IgG (IgG). (C and D) The percentage of BlaM + MDMs from multiple infections with (C) HIV-1 Lai/Balenv or (D) HIV-1 Lai/YU-2env and the means ± SEM are shown. Each symbol represents data obtained from cells derived from an independent donor. NT: untreated, IFN pretreated with 100U/ml IFN-α for 2 days. (E and F) MDMs were pretreated with the indicated doses of IFN-α for 2 days and uninfected or infected with SIVmac239 Vpx-containing VLPs, and then incubated with isotype-matched IgG (IgG) or anti-cd169 blocking antibody (αcd169) prior to infection with (E) HIV-1 Lai/Balenv-luc or (F) HIV-1 Lai/YU-2env-luc reporter virus. HIV-1 infection was quantified 3 days post infection by measuring luciferase activity in cell lysates and values were normalized to those from untreated (no IFN-α) MDMs with isotypematched IgG. The data shown are the means ± SEM of six (E) and five (F) independent experiments performed using cells from different donors. Two-tailed p values were calculated using one-way ANOVA followed by the Tukey-Kramer post test (C and D) or paired t-test (E and F) in GraphPad Prism 5. *: p 0.05, **: p 0.01, ***: p 0.001, ns: not significant. Figure 6. CD169 + cells in LNs were infected with SHIV in the presence of type I IFN signatures in vivo (A) Quantification of IP-10 mrnas in mesenteric LNs. RNA was extracted from frozen tissues which were removed from uninfected (n=5) or SHIV-infected macaques (n=5) upon necropsy and IP-10 mrna was quantitated by custom digital molecular barcoding using the NanoString ncounter System. Two-tailed p values 35

36 were calculated using unpaired t-test in GraphPad Prism 5. *: p (B) Representative images of immunofluorescence staining of mesenteric LNs of uninfected and SHIV-infected macaques. Frozen tissues were stained for CD169 (green), p27 gag (red) and nucleus (blue). The scale bar = 100 µm. (C and D) Quantitation of CD169 + cells and SHIV-infected cells (p27 gag+ ) in LNs of uninfected (n=3) and SHIV-infected macaques (n=4). CD169 + cells and p27 gag+ cells were counted by automated imaging software for 4-7 fields per LN. The percentage of (C) CD169 + cells in total cells or (D) p27 gag+ cells and double positive (CD169 + p27 gag+ ) cells in total cells was calculated and the means ± SEM are shown. Figure 7. CD169 on mature DCs attenuates the effects of ISGs in transinfection. (A and B) CD4 + T cells untreated (CF) or treated with IFN-α (CF IFNα) were infected with indicated doses of (A) X4-tropic (Lai-luc) or (B) R5-tropic HIV-1 (Lai/Balenv-luc) reporter viruses. Alternatively, IFN-DCs were pulsed with the indicated amounts of (A) X4-tropic (Lai-luc) or (B) R5-tropic (Lai/Balenv-luc) viruses, washed and cocultured with IFN-α-treated CD4 + T cells (DC-T IFNα). Infection in CD4 + T cells was quantified on 3 days post infection by measuring luciferase activity in cell lysates. The data shown are the means ± SEM of independent experiments performed in triplicates using cells from three (A) or four (B) different donors. (C) CD4 + T cells with or without IFN-α pretreatment were exposed to cell-free HIV-1 Lai-luc as in (A). Alternatively, IFN-DCs were treated with isotype-matched IgG or anti-cd169 blocking antibody prior to incubation with HIV-1, washed and added to IFN-α-treated CD4 + T cells. Infection in CD4 + T cells was quantified on 3 days post infection by measuring luciferase activity in cell lysates. The data are shown as the means ± 36

37 SEM and each symbol represents data obtained from cells derived from an independent donor. (D) CD4 + T cells were infected with cell-free virus or IFN-DCladen virus as in (C) except that HIV-1 Lai-luc containing BlaM-Vpr was used. HIV-1 fusion in CD4 + T cells was quantified at 4 hours post infection. The data are shown as the means ± SEM and each symbol represents data obtained from cells derived from an independent donor. Two-tailed p values were calculated using one sample t- test (A and B) or one-way ANOVA followed by the Tukey-Kramer post test (C and D) in GraphPad Prism 5. *: p 0.05, **: p 0.01, ***: p 0.001, ns: not significant. Downloaded from on August 16, 2018 by guest 37

38 859 References Stacey AR, Norris PJ, Qin L, Haygreen EA, Taylor E, Heitman J, Lebedeva M, DeCamp A, Li D, Grove D, Self SG, Borrow P Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections. J Virol 83: Colonna M, Trinchieri G, Liu YJ Plasmacytoid dendritic cells in immunity. Nat Immunol 5: Beignon AS, McKenna K, Skoberne M, Manches O, DaSilva I, Kavanagh DG, Larsson M, Gorelick RJ, Lifson JD, Bhardwaj N Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions. J Clin Invest 115: Fonteneau JF, Larsson M, Beignon AS, McKenna K, Dasilva I, Amara A, Liu YJ, Lifson JD, Littman DR, Bhardwaj N Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells. J Virol 78: O'Brien M, Manches O, Bhardwaj N Plasmacytoid dendritic cells in HIV infection. Adv Exp Med Biol 762: Mandl JN, Barry AP, Vanderford TH, Kozyr N, Chavan R, Klucking S, Barrat FJ, Coffman RL, Staprans SI, Feinberg MB Divergent TLR7 and TLR9 signaling and type I interferon production distinguish pathogenic and nonpathogenic AIDS virus infections. Nat Med 14: von Sydow M, Sonnerborg A, Gaines H, Strannegard O Interferonalpha and tumor necrosis factor-alpha in serum of patients in various stages of HIV-1 infection. AIDS Res Hum Retroviruses 7: Douek DC, Roederer M, Koup RA Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med 60: Hunt PW, Sinclair E, Rodriguez B, Shive C, Clagett B, Funderburg N, Robinson J, Huang Y, Epling L, Martin JN, Deeks SG, Meinert CL, Van Natta ML, Jabs DA, Lederman MM Gut Epithelial Barrier Dysfunction and Innate Immune Activation Predict Mortality in Treated HIV Infection. J Infect Dis doi: /infdis/jiu Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, Blazar BR, Rodriguez B, Teixeira-Johnson L, Landay A, Martin JN, Hecht FM, Picker LJ, Lederman MM, Deeks SG, Douek DC Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12: Estes JD, Harris LD, Klatt NR, Tabb B, Pittaluga S, Paiardini M, Barclay GR, Smedley J, Pung R, Oliveira KM, Hirsch VM, Silvestri G, Douek DC, Miller CJ, Haase AT, Lifson J, Brenchley JM Damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections. PLoS Pathog 6:e

39 Taiwo B, Barcena L, Tressler R Understanding and controlling chronic immune activation in the HIV-infected patients suppressed on combination antiretroviral therapy. Curr HIV/AIDS Rep 10: Modjarrad K, Vermund SH Effect of treating co-infections on HIV-1 viral load: a systematic review. Lancet Infect Dis 10: Doitsh G, Galloway NL, Geng X, Yang Z, Monroe KM, Zepeda O, Hunt PW, Hatano H, Sowinski S, Munoz-Arias I, Greene WC Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 505: Herbeuval JP, Nilsson J, Boasso A, Hardy AW, Kruhlak MJ, Anderson SA, Dolan MJ, Dy M, Andersson J, Shearer GM Differential expression of IFN-alpha and TRAIL/DR5 in lymphoid tissue of progressor versus nonprogressor HIV-1-infected patients. Proc Natl Acad Sci U S A 103: O'Brien M, Manches O, Sabado RL, Baranda SJ, Wang Y, Marie I, Rolnitzky L, Markowitz M, Margolis DM, Levy D, Bhardwaj N Spatiotemporal trafficking of HIV in human plasmacytoid dendritic cells defines a persistently IFN-alpha-producing and partially matured phenotype. J Clin Invest 121: Boasso A, Royle CM, Doumazos S, Aquino VN, Biasin M, Piacentini L, Tavano B, Fuchs D, Mazzotta F, Lo Caputo S, Shearer GM, Clerici M, Graham DR Overactivation of plasmacytoid dendritic cells inhibits antiviral T-cell responses: a model for HIV immunopathogenesis. Blood 118: Haller O, Kochs G Human MxA protein: an interferon-induced dynamin-like GTPase with broad antiviral activity. J Interferon Cytokine Res 31: Skaug B, Chen ZJ Emerging role of ISG15 in antiviral immunity. Cell 143: Zhao C, Collins MN, Hsiang TY, Krug RM Interferon-induced ISG15 pathway: an ongoing virus-host battle. Trends Microbiol 21: Goujon C, Moncorge O, Bauby H, Doyle T, Ward CC, Schaller T, Hue S, Barclay WS, Schulz R, Malim MH Human MX2 is an interferoninduced post-entry inhibitor of HIV-1 infection. Nature 502: Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, Florens L, Washburn MP, Skowronski J Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474: Kane M, Yadav SS, Bitzegeio J, Kutluay SB, Zang T, Wilson SJ, Schoggins JW, Rice CM, Yamashita M, Hatziioannou T, Bieniasz PD MX2 is an interferon-induced inhibitor of HIV-1 infection. Nature 502: Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, Yatim A, Emiliani S, Schwartz O, Benkirane M SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474: Liu Z, Pan Q, Ding S, Qian J, Xu F, Zhou J, Cen S, Guo F, Liang C The interferon-inducible MxB protein inhibits HIV-1 infection. Cell Host Microbe 14: Neil SJ, Zang T, Bieniasz PD Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451:

40 Sheehy AM, Gaddis NC, Choi JD, Malim MH Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418: Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, Stephens EB, Guatelli J The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3: Goujon C, Malim MH Characterization of the alpha interferon-induced postentry block to HIV-1 infection in primary human macrophages and T cells. J Virol 84: Hartnell A, Steel J, Turley H, Jones M, Jackson DG, Crocker PR Characterization of human sialoadhesin, a sialic acid binding receptor expressed by resident and inflammatory macrophage populations. Blood 97: Puryear WB, Akiyama H, Geer SD, Ramirez NP, Yu X, Reinhard BM, Gummuluru S Interferon-inducible mechanism of dendritic cellmediated HIV-1 dissemination is dependent on Siglec-1/CD169. PLoS Pathog 9:e Izquierdo-Useros N, Lorizate M, Puertas MC, Rodriguez-Plata MT, Zangger N, Erikson E, Pino M, Erkizia I, Glass B, Clotet B, Keppler OT, Telenti A, Krausslich HG, Martinez-Picado J Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides. PLoS Biol 10:e Izquierdo-Useros N, Lorizate M, Contreras FX, Rodriguez-Plata MT, Glass B, Erkizia I, Prado JG, Casas J, Fabrias G, Krausslich HG, Martinez-Picado J Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1. PLoS Biol 10:e Puryear WB, Yu X, Ramirez NP, Reinhard BM, Gummuluru S HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells. Proc Natl Acad Sci U S A 109: Crocker PR, Paulson JC, Varki A Siglecs and their roles in the immune system. Nat Rev Immunol 7: Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch A, Hiepe F, Burmester GR, Grutzkau A Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 58: York MR, Nagai T, Mangini AJ, Lemaire R, van Seventer JM, Lafyatis R A macrophage marker, Siglec-1, is increased on circulating monocytes in patients with systemic sclerosis and induced by type I interferons and tolllike receptor agonists. Arthritis Rheum 56: Jaroenpool J, Rogers KA, Pattanapanyasat K, Villinger F, Onlamoon N, Crocker PR, Ansari AA Differences in the constitutive and SIV infection induced expression of Siglecs by hematopoietic cells from nonhuman primates. Cell Immunol 250: van der Kuyl AC, van den Burg R, Zorgdrager F, Groot F, Berkhout B, Cornelissen M Sialoadhesin (CD169) expression in CD14+ cells is 40

41 upregulated early after HIV-1 infection and increases during disease progression. PLoS One 2:e Sewald X, Ladinsky MS, Uchil PD, Beloor J, Pi R, Herrmann C, Motamedi N, Murooka TT, Brehm MA, Greiner DL, Shultz LD, Mempel TR, Bjorkman PJ, Kumar P, Mothes W Retroviruses use CD169-mediated transinfection of permissive lymphocytes to establish infection. Science 350: Ambrose Z, Palmer S, Boltz VF, Kearney M, Larsen K, Polacino P, Flanary L, Oswald K, Piatak M, Jr., Smedley J, Shao W, Bischofberger N, Maldarelli F, Kimata JT, Mellors JW, Hu SL, Coffin JM, Lifson JD, KewalRamani VN Suppression of viremia and evolution of human immunodeficiency virus type 1 drug resistance in a macaque model for antiretroviral therapy. J Virol 81: Kline C, Ndjomou J, Franks T, Kiser R, Coalter V, Smedley J, Piatak M, Jr., Mellors JW, Lifson JD, Ambrose Z Persistence of viral reservoirs in multiple tissues after antiretroviral therapy suppression in a macaque RT- SHIV model. PLoS One 8:e Akiyama H, Miller C, Patel HV, Hatch SC, Archer J, Ramirez NG, Gummuluru S Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus. J Virol 88: Goujon C, Jarrosson-Wuilleme L, Bernaud J, Rigal D, Darlix JL, Cimarelli A With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIV(MAC). Gene Ther 13: Hatch SC, Archer J, Gummuluru S Glycosphingolipid composition of human immunodeficiency virus type 1 (HIV-1) particles is a crucial determinant for dendritic cell-mediated HIV-1 trans-infection. J Virol 83: Akiyama H, Ramirez NG, Gudheti MV, Gummuluru S CD169- mediated trafficking of HIV to plasma membrane invaginations in dendritic cells attenuates efficacy of anti-gp120 broadly neutralizing antibodies. PLoS Pathog 11:e Bjorndal A, Deng H, Jansson M, Fiore JR, Colognesi C, Karlsson A, Albert J, Scarlatti G, Littman DR, Fenyo EM Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 71: Cavrois M, De Noronha C, Greene WC A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20: Cline AN, Bess JW, Piatak M, Jr., Lifson JD Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. J Med Primatol 34: Polacino P, Cleveland B, Zhu Y, Kimata JT, Overbaugh J, Anderson D, Hu SL Immunogenicity and protective efficacy of Gag/Pol/Env vaccines derived from temporal isolates of SIVmne against cognate virus challenge. J Med Primatol 36: Kent KA, Gritz L, Stallard G, Cranage MP, Collignon C, Thiriart C, Corcoran T, Silvera P, Stott EJ Production and of monoclonal 41

42 antibodies to simian immunodeficiency virus envelope glycoproteins. AIDS 5: Tapias V, Greenamyre JT, Watkins SC Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro. Neurobiol Dis 54: Ambrose Z, Kline C, Polacino P, Hu SL Dysregulation of multiple inflammatory molecules in lymph node and ileum of macaques during RT- SHIV infection with or without antiretroviral therapy. J Med Primatol 43: Butler SL, Hansen MS, Bushman FD A quantitative assay for HIV DNA integration in vivo. Nat Med 7: Perreira JM, Chin CR, Feeley EM, Brass AL IFITMs restrict the replication of multiple pathogenic viruses. J Mol Biol 425: Goila-Gaur R, Strebel K HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology 5: Rempel H, Calosing C, Sun B, Pulliam L Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity. PLoS One 3:e Zou Z, Chastain A, Moir S, Ford J, Trandem K, Martinelli E, Cicala C, Crocker P, Arthos J, Sun PD Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids. PLoS One 6:e St Gelais C, de Silva S, Amie SM, Coleman CM, Hoy H, Hollenbaugh JA, Kim B, Wu L SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dntp depletion, but its expression in DCs and primary CD4+ T- lymphocytes cannot be upregulated by interferons. Retrovirology 9: Dragin L, Nguyen LA, Lahouassa H, Sourisce A, Kim B, Ramirez BC, Margottin-Goguet F Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply. Retrovirology 10: Krutzik SR, Tan B, Li H, Ochoa MT, Liu PT, Sharfstein SE, Graeber TG, Sieling PA, Liu YJ, Rea TH, Bloom BR, Modlin RL TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells. Nat Med 11: Naik SH, Metcalf D, van Nieuwenhuijze A, Wicks I, Wu L, O'Keeffe M, Shortman K Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. Nat Immunol 7: Randolph GJ, Beaulieu S, Lebecque S, Steinman RM, Muller WA Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282: Shi C, Pamer EG Monocyte recruitment during infection and inflammation. Nat Rev Immunol 11: Paquette RL, Hsu NC, Kiertscher SM, Park AN, Tran L, Roth MD, Glaspy JA Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J Leukoc Biol 64: Santini SM, Lapenta C, Logozzi M, Parlato S, Spada M, Di Pucchio T, Belardelli F Type I interferon as a powerful adjuvant for monocytederived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J Exp Med 191:

43 Bolen CR, Robek MD, Brodsky L, Schulz V, Lim JK, Taylor MW, Kleinstein SH The blood transcriptional signature of chronic hepatitis C virus is consistent with an ongoing interferon-mediated antiviral response. J Interferon Cytokine Res 33: Bosinger SE, Li Q, Gordon SN, Klatt NR, Duan L, Xu L, Francella N, Sidahmed A, Smith AJ, Cramer EM, Zeng M, Masopust D, Carlis JV, Ran L, Vanderford TH, Paiardini M, Isett RB, Baldwin DA, Else JG, Staprans SI, Silvestri G, Haase AT, Kelvin DJ Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys. J Clin Invest 119: Harris LD, Tabb B, Sodora DL, Paiardini M, Klatt NR, Douek DC, Silvestri G, Muller-Trutwin M, Vasile-Pandrea I, Apetrei C, Hirsch V, Lifson J, Brenchley JM, Estes JD Downregulation of robust acute type I interferon responses distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection of natural hosts from pathogenic SIV infection of rhesus macaques. Journal of Virology 84: Jacquelin B, Mayau V, Targat B, Liovat AS, Kunkel D, Petitjean G, Dillies MA, Roques P, Butor C, Silvestri G, Giavedoni LD, Lebon P, Barre- Sinoussi F, Benecke A, Muller-Trutwin MC Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response. The Journal of clinical investigation 119: Sandler NG, Bosinger SE, Estes JD, Zhu RT, Tharp GK, Boritz E, Levin D, Wijeyesinghe S, Makamdop KN, del Prete GQ, Hill BJ, Timmer JK, Reiss E, Yarden G, Darko S, Contijoch E, Todd JP, Silvestri G, Nason M, Norgren RB, Jr., Keele BF, Rao S, Langer JA, Lifson JD, Schreiber G, Douek DC Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression. Nature 511: Sedaghat AR, German J, Teslovich TM, Cofrancesco J, Jr., Jie CC, Talbot CC, Jr., Siliciano RF Chronic CD4+ T-cell activation and depletion in human immunodeficiency virus type 1 infection: type I interferonmediated disruption of T-cell dynamics. Journal of Virology 82: Wilson EB, Yamada DH, Elsaesser H, Herskovitz J, Deng J, Cheng G, Aronow BJ, Karp CL, Brooks DG Blockade of chronic type I interferon signaling to control persistent LCMV infection. Science 340: Teijaro JR, Ng C, Lee AM, Sullivan BM, Sheehan KC, Welch M, Schreiber RD, de la Torre JC, Oldstone MB Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340: Deeks SG, Tracy R, Douek DC Systemic effects of inflammation on health during chronic HIV infection. Immunity 39: Doyle T, Goujon C, Malim MH HIV-1 and interferons: who's interfering with whom? Nat Rev Microbiol doi: /nrmicro Feeley EM, Sims JS, John SP, Chin CR, Pertel T, Chen LM, Gaiha GD, Ryan BJ, Donis RO, Elledge SJ, Brass AL IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog 7:e Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, Adams DJ, Xavier RJ, Farzan M, Elledge SJ The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:

44 Lu J, Pan Q, Rong L, He W, Liu SL, Liang C The IFITM proteins inhibit HIV-1 infection. J Virol 85: Hladik F, McElrath MJ Setting the stage: host invasion by HIV. Nat Rev Immunol 8: Fenton-May AE, Dibben O, Emmerich T, Ding H, Pfafferott K, Aasa- Chapman MM, Pellegrino P, Williams I, Cohen MS, Gao F, Shaw GM, Hahn BH, Ochsenbauer C, Kappes JC, Borrow P Relative resistance of HIV-1 founder viruses to control by interferon-alpha. Retrovirology 10: Parrish NF, Gao F, Li H, Giorgi EE, Barbian HJ, Parrish EH, Zajic L, Iyer SS, Decker JM, Kumar A, Hora B, Berg A, Cai F, Hopper J, Denny TN, Ding H, Ochsenbauer C, Kappes JC, Galimidi RP, West AP, Jr., Bjorkman PJ, Wilen CB, Doms RW, O'Brien M, Bhardwaj N, Borrow P, Haynes BF, Muldoon M, Theiler JP, Korber B, Shaw GM, Hahn BH Phenotypic properties of transmitted founder HIV-1. Proc Natl Acad Sci U S A 110: Deymier MJ, Ende Z, Fenton-May AE, Dilernia DA, Kilembe W, Allen SA, Borrow P, Hunter E Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-alpha Resistance. PLoS Pathog 11:e Etemad B, Gonzalez OA, White L, Laeyendecker O, Kirk GD, Mehta S, Sagar M Characterization of HIV-1 envelopes in acutely and chronically infected injection drug users. Retrovirology 11: Oberle CS, Joos B, Rusert P, Campbell NK, Beauparlant D, Kuster H, Weber J, Schenkel CD, Scherrer AU, Magnus C, Kouyos R, Rieder P, Niederost B, Braun DL, Pavlovic J, Boni J, Yerly S, Klimkait T, Aubert V, Trkola A, Metzner KJ, Gunthard HF, Swiss HIVCS Tracing HIV-1 transmission: envelope traits of HIV-1 transmitter and recipient pairs. Retrovirology 13: Berger EA, Murphy PM, Farber JM Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 17: Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR Change in coreceptor use correlates with disease progression in HIV-1--infected individuals. J Exp Med 185: Ince WL, Zhang L, Jiang Q, Arrildt K, Su L, Swanstrom R Evolution of the HIV-1 env gene in the Rag2-/- gammac-/- humanized mouse model. J Virol 84: Ren W, Tasca S, Zhuang K, Gettie A, Blanchard J, Cheng-Mayer C Different tempo and anatomic location of dual-tropic and X4 virus emergence in a model of R5 simian-human immunodeficiency virus infection. J Virol 84: Jayakumar P, Berger I, Autschbach F, Weinstein M, Funke B, Verdin E, Goldsmith MA, Keppler OT Tissue-resident macrophages are productively infected ex vivo by primary X4 isolates of human immunodeficiency virus type 1. J Virol 79: Verani A, Pesenti E, Polo S, Tresoldi E, Scarlatti G, Lusso P, Siccardi AG, Vercelli D CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates. J Immunol 161:

45 Martin-Garcia J, Cao W, Varela-Rohena A, Plassmeyer ML, Gonzalez- Scarano F HIV-1 tropism for the central nervous system: Brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor. Virology 346: McDonald D, Wu L, Bohks SM, KewalRamani VN, Unutmaz D, Hope TJ Recruitment of HIV and its receptors to dendritic cell-t cell junctions. Science 300: Felts RL, Narayan K, Estes JD, Shi D, Trubey CM, Fu J, Hartnell LM, Ruthel GT, Schneider DK, Nagashima K, Bess JW, Jr., Bavari S, Lowekamp BC, Bliss D, Lifson JD, Subramaniam S D visualization of HIV transfer at the virological synapse between dendritic cells and T cells. Proc Natl Acad Sci U S A 107: Dale BM, McNerney GP, Thompson DL, Hubner W, de Los Reyes K, Chuang FY, Huser T, Chen BK Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion. Cell Host Microbe 10: Kaplan ME, Coons AH, Deane HW Localization of antigen in tissue cells; cellular distribution of pneumococcal polysaccharides types II and III in the mouse. J Exp Med 91:15-30, 14 pl. 97. Nossal GJ, Abbot A, Mitchell J Antigens in immunity. XIV. Electron microscopic radioautographic studies of antigen capture in the lymph node medulla. J Exp Med 127: Nossal GJ, Abbot A, Mitchell J, Lummus Z Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J Exp Med 127: Iannacone M, Moseman EA, Tonti E, Bosurgi L, Junt T, Henrickson SE, Whelan SP, Guidotti LG, von Andrian UH Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus. Nature 465: Junt T, Moseman EA, Iannacone M, Massberg S, Lang PA, Boes M, Fink K, Henrickson SE, Shayakhmetov DM, Di Paolo NC, van Rooijen N, Mempel TR, Whelan SP, von Andrian UH Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450: Carrasco YR, Batista FD B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27: Phan TG, Green JA, Gray EE, Xu Y, Cyster JG Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat Immunol 10: Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: Honke N, Shaabani N, Cadeddu G, Sorg UR, Zhang DE, Trilling M, Klingel K, Sauter M, Kandolf R, Gailus N, van Rooijen N, Burkart C, Baldus SE, Grusdat M, Lohning M, Hengel H, Pfeffer K, Tanaka M, Haussinger D, Recher M, Lang PA, Lang KS Enforced viral 45

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