Nef-Mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication Are Fundamental Properties of Primate Lentiviruses

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1 JOURNAL OF VIROLOGY, Dec. 2007, p Vol. 81, No X/07/$ doi: /jvi Copyright 2007, American Society for Microbiology. All Rights Reserved. Nef-Mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication Are Fundamental Properties of Primate Lentiviruses Jan Münch, 1 Devi Rajan, 1 Michael Schindler, 1 Anke Specht, 1 ElkeRücker, 1 Francis J. Novembre, 2 Eric Nerrienet, 3 Michaela C. Müller-Trutwin, 4 Martine Peeters, 5 Beatrice H. Hahn, 6 and Frank Kirchhoff 1 * Institute of Virology, University of Ulm, Ulm, Germany 1 ; Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia ; Centre Pasteur du Cameroun, BP1274, Yaounde, Cameroon 3 ; Unité de Biologie des Rétrovirus, Institut Pasteur, Paris, France 4 ; Departments of Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, Alabama ; and Laboratoire Retrovirus, UMR145, Institut de Recherche pour le Development and Department of International Health, University of Montpellier, Montpellier, France 6 Received 27 April 2007/Accepted 26 September 2007 Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-cd3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans. Downloaded from The accessory nef gene is present in the genomes of all primate lentiviruses but absent in other retroviruses. Early studies have established that Nef accelerates progression to AIDS and is required for efficient viral replication and persistence of human immunodeficiency virus type 1 (HIV-1) in humans (16, 36) and of the simian immunodeficiency virus SIVmac in experimentally infected rhesus macaques (35). Hence, Nef has been characterized as a key factor of primate lentiviral pathogenicity (75). Subsequently, a striking number of Nef functions and interactions have been identified, including downregulation of CD4, CD28, and class I major histocompatibility complex (MHC-I); enhancement of virion infectivity; stimulation of viral replication; and alteration of T-cell activation pathways (reviewed in references 3, 14, 33, 56, 57, and 75). Accumulating evidence suggests that the combination of these Nef activities is important for efficient viral persistence and accelerated disease progression in HIV-1-infected humans and in SIVmac-infected macaques (7, 10, 19, 26, 32, 49, 60, 70). * Corresponding author. Mailing address: Institute of Virology, University of Ulm, Albert Einstein Allee 11, D Ulm, Germany. Phone: Fax: J.M., D.R., M.S., and A.S. contributed equally to this work. Present address: Heinrich Pette Institut, Hamburg, Germany. Present address: HIV/Hepatitis Laboratory, Institut Pasteur du Cambodge, BP983, Phnom Penh, Cambodia. Published ahead of print on 10 October For a long time, our knowledge about Nef function was mainly derived from the analysis of HIV-1 and, to a much lesser extent, HIV-2 and SIVmac nef alleles. Recently, however, it has been shown that some Nef activities, i.e., the ability to downmodulate CD4, CD28, and MHC-I, are conserved between most or all lineages of primate lentiviruses (62). In contrast, nef alleles from various lineages of HIV and SIV differ fundamentally in their ability to downregulate CD3, a key component of the T-cell receptor (TCR) complex. HIV-2 and the majority of SIV nef alleles downmodulate TCR-CD3 with high efficiency, thereby suppressing the responsiveness of virally infected T cells to stimulation and activation-induced cell death (6, 30, 62). In contrast, Nef proteins from HIV-1 and its closest simian relatives from chimpanzees and some Cercopithecus monkeys generally failed to downregulate TCR-CD3 (62). Notably, inefficient downmodulation of TCR-CD3 was associated with low CD4 T-cell counts in SIVsmm-infected sooty mangabeys (62), suggesting that this Nef function plays a protective role in natural SIV infection in vivo. The analysis of receptor modulation by different primate lentiviral Nef proteins revealed that the loss of one specific Nef function in the lineage that gave rise to HIV-1 may partly explain the different levels of immune activation observed in pathogenic and nonpathogenic HIV and SIV infections (62). Possible lineage-specific differences in other Nef functions remained to be investigated. For example, it is well established that the HIV-1 Nef enhances virion infectivity and stimulates viral replication in primary human T cells and in human lym- on August 17, 2018 by guest 13852

2 VOL. 81, 2007 Nef-MEDIATED ENHANCEMENT OF VIRAL INFECTIVITY FIG. 1. Generation of HIV-1 NL4-3 constructs expressing heterologous HIV and SIV nef alleles. Restriction sites used to clone heterologous HIV and SIV nef alleles into the HIV-1 NL4-3 IRES-eGFP (61, 62) and HIV-1 NL4-3nef 1 2 (46) constructs are indicated. Critical cis-regulatory elements encompassing the TPI region as well as the start of the LTR region and the position of the 266-bp U3 deletion downstream of nef in the NL4-3nef 1 2 clones (46) are shown at the bottom. phoid tissue (HLT) ex vivo (1, 2, 12, 25, 45, 65, 68). Infectivity enhancement requires expression of Nef in the virus-producing cell and involves an early step of the viral replication cycle (1, 65). The mechanism is currently controversial and may involve reduced susceptibility of the viral particles to proteasomal degradation (55), facilitated transport of the viral genome through the cortical actin network (9), and/or increased cholesterol content of progeny virions (78). Notably, the ability of Nef to enhance virion infectivity does not correlate with its capacity to promote HIV-1 replication (26, 39). Nef efficiently enhances HIV-1 replication only in primary T-cell cultures and in ex vivo infected HLT but not in transformed T-cell lines (3, 68). It has been suggested that Nef-mediated CD4 downmodulation (26, 39) and/or activation of resting T cells (2, 19, 64, 74) contributes to its enhancing effect on viral replication. Importantly, studies in the SIV/macaque model and the functional analysis of nef alleles from long-term survivors of HIV-1 infection suggest that both Nef activities are relevant for efficient viral persistence and the pathogenesis of AIDS in vivo (7, 10, 15, 19, 32, 41, 73). Differences in Nef-mediated enhancement of virion infectivity and stimulation of viral replication could potentially affect the virulence, persistence, and transmission of various primate lentiviruses. It has been shown that these two HIV-1 Nef activities are also conserved in HIV-2 and SIVsmm nef alleles, although HIV-2 Nefs are usually less active than those of HIV-1 (48). However, it is currently unknown whether nef alleles from other primate lentiviruses, including those of SIVcpz, the simian counterpart of HIV-1 (23, 66), are also capable of performing these functions. Here, we show that the great majority of nef alleles from 15 different primate lentiviruses enhance viral infectivity and replication in human cells or tissues. Unpassaged SIVcpz and SIVsmm nef alleles were active in these assays, suggesting that no adaptive changes were required after cross-species transmission from chimpanzees or sooty mangabeys, respectively, to humans. Furthermore, nef alleles from several highly divergent SIVs that have not been found in humans also enhanced viral replication and CD4 T-cell depletion in ex vivo infected HLT, supporting the idea that lack of Nef function does not usually represent a barrier for zoonotic transmissions of primate lentiviruses from monkeys to humans. MATERIALS AND METHODS Proviral constructs. The pbr-nl43-ires-egfp-nef construct was generated by cloning an internal ribosome entry site (IRES) and the gene encoding the enhanced version of the green fluorescent protein (egfp) into pbr-nl4-3nef 1 2 (46) (Fig. 1). Firstly, an HpaIHI/XbaI restriction fragment of pbr- NL4-3nef 1 2 encompassing the nef gene and the 3 long terminal repeat (LTR) was cloned into pcrii-topo (Invitrogen, Carlsbad, CA) to generate pcrii-topo-nef 1 2. Secondly, we amplified by PCR the egfp gene of pegfp-c1 (Clontech Laboratories, Inc.) using primers introducing flanking SmaI/NheI and SmaI sites. This PCR fragment was cloned downstream of nef

3 13854 MÜNCH ET AL. J. VIROL. into pcrii-topo-nef 1 2. Thirdly, the IRES element of pcgcg-nef-ires- GFP was inserted between nef and egfp using the MluI and NcoI restriction sites. In the last step, the nef-ires-egfp-ltr fragment was cloned into pbr- NL4-nef 1 2 to generate pbr-nl43-ires-egfp-nef. Derivatives containing stop codons at positions 73 and 74 of the nef open reading frame (ORF) either alone (nef*) or in combination with mutations in the ATG initiation codon and two in-frame stop codons at positions four and five of the nef ORF (nef ) disrupting the NL4-3 nef gene were generated by standard PCR and cloning techniques. Splice-overlap extension PCR was used to replace the NL4-3 nef gene with a large number of heterologous HIV and SIV nef alleles (62). Briefly, PCR fragments containing the 3 end of the NL4-3 env gene fused to the respective nef genes was cloned into pbr-nl43-ires-egfp-nef using the unique HpaI and MluI sites. Most HIV-1 NL4-3 IRES-eGFP nef constructs used in this study are available through the NIH AIDS Research and Reference Reagent Program (ARRRP). To generate NL4-3 nef constructs without an IRES-eGFP element, env-nef fragments derived from the NL4-3-based Nef- IRES-eGFP constructs (62) were cloned into HIV-1 NL4-3nef 1 2 (46) using the single HpaI and MluI restrictions sites in env and just downstream of the nef gene, respectively (Fig. 1). As indicated in Fig. 1, HIV-1 NL4-3 TPI nef variants contain intact critical cis-regulatory elements, encompassing the T-rich region, polypurine tract, and attachment (att) sequences required for integration (herein after referred to as the TPI region) downstream of the respective nef alleles. HIV-1 NL4-3 nef recombinants containing the V3 loop region of the CCR5- tropic HIV-1 92TH014-2 strain were generated by standard cloning and PCR mutagenesis techniques as described previously (47, 51). Cell culture and virus stocks. P4-CCR5, TZM-bl, and 293T cells were cultured as described previously (17, 47, 48). Briefly, TZM-bl cells (53, 75) were obtained from the NIH ARRRP, as contributed by John Kappes and Xiaoyun Wu. P4- CCR5 cells are a derivative of P4 cells (11) expressing the CCR5 receptor (21). These cells and 293T cells were maintained in Dulbecco s modified Eagle s medium containing 10% heat-inactivated fetal bovine serum. Peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated using lymphocyte separation medium (Biocoll separating solution; Biochrom), stimulated for 3 days with phytohemagglutinin (2 g/ml), and cultured in RPMI 1640 medium with 10% fetal calf serum and 10 ng/ml interleukin-2 (IL-2) prior to infection. For the generation of virus stocks, 293T cells were transfected by the calcium phosphate method with proviral constructs as described previously (48, 62). The medium was changed after overnight incubation, and virus was harvested 24 h later. Residual cells in the supernatants were pelleted, and the supernatants were stored at 70 C. The content of viral p24 antigen was quantified by an HIV p24 enzyme-linked immunosorbent assay kit obtained through the NIH ARRRP. Infectivity assays. Virus infectivity was determined using TZM-bl and P4- CCR5 cells as described previously (47, 48). Briefly, the cells were sown out in 96-well dishes in a volume of 100 l and infected after overnight incubation with virus stocks containing 1 or 5 ng of p24 antigen produced by transiently transfected 293T cells. Two days postinfection viral infectivity was detected using a galactosidase screen kit from Tropix as recommended by the manufacturer. -Galactosidase activities were quantified as relative light units per second using an Orion Microplate Luminometer. To calculate percent values, relative light units per second obtained for wild-type HIV-1 NL4-3 infection were set to 100%. Viral replication in PBMC cultures. The efficiency of HIV-1 replication in PBMCs was determined as described previously (47). Briefly, unstimulated PBMCs were sown out in 150 l of medium and infected with virus stocks containing 1 ng of p24 antigen in a total volume of 200 l. At 3 days postinfection the cells were stimulated with phytohemagglutinin (2 g/ml) and 10 ng/ml IL-2 for 2 days. Thereafter, PBMCs were cultivated in RPMI medium containing 10 ng/ml IL-2. Supernatants were harvested at regular intervals, and fresh medium was added. The extent of viral replication was determined by reverse transcriptase (RT) assay as described previously (54). All experiments were performed at least in duplicate with two independent virus stocks. Ex vivo human lymphoid tissue. Human tonsillar tissue removed during routine tonsillectomy and not required for clinical purposes was received within 5 h of excision. The tonsils were washed thoroughly with medium containing antibiotics and then sectioned into 2- to 3-mm 3 blocks. These tissue blocks were placed on top of collagen sponge gels in the culture medium at the air-liquid interface and infected as described previously (24, 59). A total of 10 l of virus stock containing 0.5 ng of p24 antigens was applied to each tissue block. Supernatants were collected at 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. Flow cytometry was performed on cells mechanically isolated from control and infected tissue blocks, and depletion of CD4 T cells was quantified as described previously (24, 25, 59). For determination of the ratio of CD4 to CD8 T cells, cells were stained for surface markers by using anti-cd3 fluorescein isothiocyanate, anti-cd4-allophycocyanin, and anti-cd8 tricolor stain. In experiments including HIV-1 nef variants that downmodulate TCR-CD3, anti-cd2 instead of anti-cd3 fluorescein isothiocyanate was used. Nucleotide sequence accession numbers. The nef sequences analyzed in this study have been previously deposited in the GenBank database and can be retrieved by using the following accession numbers (strain designations are in parentheses): M19921 (NL4-3), DQ (NA7), AF (032an-93), AF (039 nm-94), M38429 (JRCSF), AY (MVP8161), AY (MVP13127), AJ (YBF30), AY (YBF116), AY (US), AY (Cam3k1), AY (Cam3k5), AY (Cam5k2), AF (GAB2), AY (Nok5), AY (ch-nik4), EF (TAN1.910), DQ (TAN2.69), DQ (TAN3.1), AF (SIVgsn CM166), AY (SIVmus CMS1085), AY (SIVmon CML1), M30502 (HIV-2 BEN), DQ (HIV-2 CBL-23), DQ (HIV K), DQ (HIV ), DQ (SIVsmm FMm1), DQ (SIVsmm FYr1), DQ (SIVsmm FWr1), M33262 (SIVmac239), AF (SIVrcm GB1), AY (SIVdeb CM5), AY (SIVdeb CM40), DQ (SIVsyk KE44), AY (SIVsyk KE51), DQ (SIVblu KE31), DQ (SIVsun sol-36), AF (SIVtan 1), DQ (SIVtan B87-18), and U04005 (SIVsab 1). Statistical analysis. The mean activities of different groups of nef alleles were compared using a Student s t test. Similar results were obtained with the Mann- Whitney test. The PRISM package, version 4.0 (Abacus Concepts, Berkeley, CA), was used for all calculations. RESULTS Nef-mediated enhancement of virion infectivity is a fundamental property of primate lentiviruses. We have recently used HIV-1 NL4-3-based proviral constructs coexpressing egfp and Nef (Fig. 1) to analyze the effect of nef alleles from different groups of primate lentiviruses on various cellular receptors and on T-cell activation and programmed cell death (62). Here, we utilized this collection of reporter viruses expressing nef genes from HIV-1 (groups M, N and O), HIV-2, the simian counterparts of both human viruses (SIVcpz and SIVsmm, respectively) (22, 23, 28, 29, 34, 66), the descendants of those SIVs that likely recombined to become SIVcpz (SIVrcm and SIVgsn/mus/mon, respectively) (5), and lentiviruses from seven additional monkey species (SIVmac, SIVdeb, SIVsyk, SIVblu, SIVsun, SIVagm.tan, and SIVagm.sab) to assess whether the ability to enhance virion infectivity is a conserved property of primate lentiviral Nef proteins. To quantify the infectivity of these HIV-1 constructs, we exposed the HeLa-CD4/LTR-lacZ indicator cell line, P4- CCR5, which expresses CD4 and both major entry cofactors CXCR4 and CCR5 (17, 21), to 293T cell-derived virus stocks containing 5 ng of p24 antigen and determined the -galactosidase activities 2 days later. We found that HIV-1 NL4-3, containing the intact wild-type nef gene, was 16.1-fold more infectious than the otherwise isogenic nef* control construct, containing a premature stop codon at position 40 of the NL4-3 nef ORF, and 5.7-fold more infectious than NL4-3 nef, containing this same stop codon combined with a second one at position 3 and a mutation in the initiation codon, respectively (Fig. 2A, nef* and nef, respectively). This two- to threefold difference between the two nef-defective control constructs was highly reproducible and suggests that expressing no Nef is less disadvantageous for HIV-1 infectivity than producing a truncated inactive form. More importantly, this analysis showed that 27 of the 30 nef alleles analyzed (90%) enhanced virion infectivity, albeit with pronounced differential efficiency ranging from 2.7- to 36.4-fold compared to the nef control virus

4 VOL. 81, 2007 Nef-MEDIATED ENHANCEMENT OF VIRAL INFECTIVITY FIG. 2. The ability of Nef to enhance the infectivity of HIV-1 IRES-eGFP reporter viruses is conserved among different groups of primate lentiviruses. (A) P4-CCR5 indicator cells were infected with HIV-1 NL4-3 IRES-eGFP constructs containing the indicated HIV and SIV nef genes or defective nef alleles. Infections were performed in triplicate with three different virus stocks containing 5 ng of p24 antigen. Shown are average values of the nine measurements SDs compared to the infectivity of the virus expressing wild-type NL4-3 Nef (100%). The graph of values for sources of the various Nef alleles is color-coded as follows: black bars HIV-1 and HIV-2; gray bars, the simian counterparts SIVcpz and SIVsmm; blue bars, descendants of the viruses that recombined to become SIVcpz; white bars, all other SIVs. The lower dotted line indicates the infectivity of the nef-defective (nef )control virus and the upper dotted line indicates the HIV-1 construct expressing the control wild-type NL4-3 nef allele. (B) Correlation between Nef-mediated enhancement of viral infectivity in P4-CCR5 and TZM-bl cells. (C) Enhancement of virion infectivity by nef alleles that do (group 2, green) or do not (group 1, red) downmodulate TCR-CD3. Average infectivities ( SEM) of HIV-1 IRES-eGFP constructs expressing nef alleles derived from HIV-1, SIVcpz, SIV-CP, HIV-2, SIVsmm, and all other SIVs (SIV AO). The numbers above the bars in panels A and C indicate the relative infectivity enhancement (n-fold) compared to the nef control virus. Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii. (Fig. 2A). Unfortunately, no antibodies are available against most of these highly divergent HIV and SIV Nef alleles. Thus, it remains to be clarified to what extent differences in the potency of infectivity enhancement may be due to different Nef expression levels. Unexpectedly, some SIV Nefs, e.g., those of SIVgsn CM166, SIVsmm FMm1, and SIVdeb CM5, were substantially more active than HIV-1 nef alleles (29.1- to 36.4-fold compared to 4.7- to 15.0-fold, respectively). To verify the differential ability of these 30 HIV and SIV nef alleles in enhancing viral infectivity, we used a second indicator cell line, TZMbl, previously designated JC53-bl (17, 76). TZM-bl cells contain both the luciferase and -galactosidase genes under the control of the HIV-1 promoter and stably express high levels of CD4 and CCR5. They are more susceptible to HIV-1 infection than P4-CCR5 cells, but the magnitude of Nef-mediated infectivity enhancement is usually lower (data not shown). This analysis confirmed that all Nef alleles that enhanced HIV-1 infectivity in P4-CCR5 cells also did so with similar potency in TZM-bl cells (Fig. 2B). These results showed that Nef-mediated infectivity enhancement is a highly conserved property of primate lentiviruses. Next, we examined possible quantitative differences in the ability of Nef proteins to enhance virion infectivity based on their biological properties, species origin, or phylogenetic relationship. Firstly, we divided the proviral constructs into those that fail (group 1 Nefs, HIV-1 and its closest simian relatives, SIVcpz and SIVgsn/mus/mon; n 13) and those that are capable of TCR-CD3 down-modulation (group 2 Nefs, HIV-2 and all other SIVs analyzed; n 17) (62). This analysis showed that group 1 and group 2 Nefs do not differ significantly in their ability to enhance viral infectivity ( versus ; P 0.83; values are average infectivities standard errors of the means [SEM] compared to the wild-type NL4-3 Nef) (Fig. 2C). Secondly, we compared the functional activities

5 13856 MÜNCH ET AL. J. VIROL. of nef alleles from HIV-1 (n 5) and HIV-2 (n 4) with those of their respective simian counterparts, SIVcpz (n 4) and SIVsmm (n 3). On average, HIV-1 and SIVcpz Nefs showed similar potency levels in enhancing viral infectivity ( versus ; P 0.73) (Fig. 2C). Two of the four HIV-2 nef alleles analyzed (CBL-23 and 60415K) were inactive in enhancing virion infectivity (Fig. 2A and B), although they efficiently downmodulated CD4 and MHC-I (48). In agreement with our previous results obtained using SIV mac239-based nef recombinants (48), the HIV-2 BEN Nef was highly active, and the Nef displayed an intermediate phenotype in infectivity assays. The activity of SIVsmm Nefs was also highly variable, but on average they enhanced viral infectivity more efficiently than those of HIV-2 ( versus ; P 0.29) (Fig. 2C). The SIVcpz GAB2cl48 (PBMC DNA), chnok5 (plasma RNA), and TAN1 (fecal RNA) nef alleles and the SIVsmm FMm1, FYr1, and FWr1 nef alleles (all PBMC DNA) were derived from unpassaged chimpanzee or sooty mangabey material, respectively (37, 48, 72). Thus, no adaptive changes were required for effective SIVcpz- and SIVsmm-mediated infectivity enhancement in human cells. Thirdly, we examined nef alleles from the descendants of those monkey viruses, i.e., SIVrcm and the precursor of closely related Cercopithecus viruses (SIVgsn/mus/ mon), that gave rise to SIVcpz (5). We found that nef alleles from SIVrcm, SIVgsn, SIVmus, and SIVmon, named SIVcpz precusors (SIV-CP), are all highly active in enhancing virion infectivity in human-derived cells (Fig. 2A and C). Finally, nef genes derived from SIVcpz and SIVsmm, which have been transmitted to humans, did not display higher functional activity in human-derived cells than those from SIVs that have not been found in humans (named SIV-AO for all others in Fig. 2C). Thus, although some HIV and SIV Nefs show only 30% homology on the amino acid level (data not shown), the functional interactions required for infectivity enhancement seem to be conserved in different primate species. The HIV-1 IRES-eGFP constructs express Nef via the wildtype HIV-1 LTR promoter and naturally occurring splice sites (61 63). However, their genomic size is enlarged, and the NL4-3 IRES-eGFP virus shows lower infectivity than the parental clone (data not shown). Moreover, the IRES-eGFP element is not advantageous for the virus and was deleted after multiple rounds of replication. Thus, these constructs are highly useful to analyze the effect of Nef on surface expression of various receptors (62, 63) and in single-cycle infection assays but less suitable to examine its ability to promote viral replication. Therefore, we next generated and analyzed HIV-1 NL4-3 constructs expressing various HIV and SIV nef alleles without an IRES-eGFP element by inserting env-nef fragments derived from the NL4-3-based Nef-IRES-eGFP constructs into HIV-1 NL4-3nef 1 2 (Fig. 1). Notably, this set included a larger number of SIVcpz nef alleles than the collection of IRES-eGFP viruses described above (10 versus 4). Since the HIV-1 genome lost the nef-env overlap typical for all other primate lentiviruses (except for SIVcpz) during primate lentiviral evolution (62), we specifically replaced the NL4-3 nef gene by genes of other viruses. To circumvent possible differences due to variations in important cis regulatory sequences in the nef ORF, all constructs contained the wild-type NL4-3 TPI region downstream of Nef and upstream of the core enhancer elements (46) (Fig. 1). Importantly, all proviral HIV-1 vectors were isogenic except for their nef coding sequences. Infection of P4-CCR5 cells with these HIV-1 constructs showed that all 29 nef alleles analyzed amplified virion infectivity between 2.3- and 21.9-fold (Fig. 3A). For the 24 nef alleles analyzed in both proviral backbones, there was a highly significant correlation between the efficiency of Nef-mediated infectivity in the context of HIV-1 constructs with and without the IRES-eGFP element (Fig. 3B). However, some nef alleles that did not significantly enhance infectivity of IRES-GFP viruses, i.e., those of HIV K and , displayed weak but reproducible enhancing effects in the wild-type NL4-3 backbone (Fig. 3A), although they were expressed from the same promoter and splice sites. One possible explanation is that the wild-type Nef-coding RNAs might be more stable than those containing the IRES-eGFP cassette and that the activity of weak Nef alleles may be detectable only under optimal expression conditions. The results obtained using the HIV-1 TPI nef recombinants confirmed that the SIVgsn CM166, SIVsmm FMm1, and SIVdeb CM40 Nefs enhance viral infectivity more efficiently than those of HIV-1 NL4-3 and NA7 (Fig. 3A). They also verified that group 1 and group 2 Nefs promote virion infectivity equally well and that HIV-1 and HIV-2 Nefs do not amplify viral infectivity in human-derived cells more efficiently than those of their simian precursors or other SIVs (Fig. 3C). Finally, the five nef alleles from SIVcpzPts (for SIVcpz from Pan troglodytes schweinfurthii) that has not been found in humans were as active as those from SIVcpzPtt (for SIVcpz from P. t. troglodytes) which has been transmitted to humans at least two or three times (Fig. 3A and C) (28, 34, 66). Thus, ineffective Nef-mediated enhancement of virion infectivity does not explain why SIVcpz from the P. t. schweinfurthii subspecies of chimpanzees has not been found in humans. It is well established that HIV-1 strains present prior to AIDS progression and the great majority of SIVs utilize CCR5 (R5) but not CXCR4 (X4) for viral entry. To determine whether the viral coreceptor tropism may affect Nef-mediated enhancement of virion infectivity, we generated a set of NL4-3 proviral constructs expressing 15 different HIV and SIV nef alleles and containing the V3 loop region of the R5-tropic 92TH014-2 HIV-1 strain in their env genes. We have previously shown that this HIV-1 NL4-3 V3 recombinant efficiently uses R5 but not X4 for viral entry (51). Our results showed that the 15 HIV-1, HIV-2, and SIV nef alleles enhanced R5-tropic HIV-1 infection of P4-CCR5 cells with efficiencies ranging from 9.6- to 71.2-fold compared to the nef control virus (Fig. 4A, nef ). The infectivities of the R5-tropic HIV-1 Nef variants correlated very well with those of their X4-tropic counterparts (Fig. 4B), suggesting that the effect of HIV and SIV nef alleles on virion infectivity is not affected by the viral coreceptor tropism. Most primate lentiviral Nefs promote viral replication in human PBMCs. It has been established that the ability of Nef to enhance virion infectivity does not correlate with its potency in stimulating viral replication in primary T cells (26, 39). Thus, both represent independent Nef functions. To assess whether the ability of Nef to stimulate viral replication is a conserved property of primate lentiviruses, we infected human PBMCs with X4-tropic HIV-1 NL4-3 constructs expressing 42 different

6 VOL. 81, 2007 Nef-MEDIATED ENHANCEMENT OF VIRAL INFECTIVITY FIG. 3. Various primate lentiviral Nef proteins increase HIV-1 infectivity. (A) P4-CCR5 cells were infected with recombinant HIV-1 constructs expressing the indicated nef alleles. Infections were performed in triplicate (each) with three independent virus stocks. (B) Correlation between Nef-mediated enhancement of NL4-3 TPI and IRES-eGFP constructs. (C) Average infectivities ( SD) of HIV-1 TPI constructs expressing nef alleles derived from the indicated groups of primate lentiviruses (see legend of Fig. 1 for details). Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii; PPT, polypurine tract. AO, all other. HIV and SIV nef alleles. We found that all recombinant viruses containing intact HIV-1 M, N, and O as well as SIVcpzPtt or SIVcpzPts nef genes replicated with higher efficiency than the nef-defective virus (Fig. 5A). Thus, nef genes from all three groups of HIV-1 and their simian counterparts are able to stimulate viral replication in human T cells, albeit with different efficiencies. On average, nef alleles from HIV-1 groups O (n 4) and N (n 2), as well as those from SIVcpzPtt (n FIG. 4. Primate lentiviral Nef proteins enhance HIV-1 infectivity independently of the viral coreceptor tropism. (A) P4-CCR5 cells were infected with recombinant R5-tropic HIV-1 constructs expressing the indicated nef alleles. Infections were performed in triplicate (each) with three independent virus stocks. Numbers above the bars indicate the relative infectivity enhancement (n-fold) compared to the nef control virus. (B) Correlation between the infectivities of X4- and R5-tropic HIV-1 nef recombinants. PPT, polypurine tract; Ptt, P. t. troglodytes; Pts, P. t. schweinfurthii.

7 13858 MÜNCH ET AL. J. VIROL. Downloaded from FIG. 5. HIV-1 Group M, N, and O and SIVcpzPtt and SIVcpzPts nef alleles enhance viral replication in human PBMCs with comparable efficiencies. (A) Representative replication kinetics of wild-type NL4-3 and nef recombinants. PBMCs were infected with HIV-1 TPI variants containing the indicated nef genes or a disrupted nef codon immediately after isolation and stimulated 3 days later. Virus production was monitored by RT assay. PSL, photon-stimulated luminescence. (B) Average levels of replication of HIV-1 NL4-3 recombinants expressing HIV-1 group M, N, and O or SIVcpz nef alleles. Results were derived from duplicate infections of two independent virus stocks. Similar results were obtained with cells derived from a different PBMC donor, except that the levels of replication in the absence of Nef were slightly higher and reached about 20% of that of wild-type NL4-3. (C) Average virus production in infected PBMC cultures. PBMCs were inoculated with the indicate NL4-3 nef recombinants, and cumulative production of p24 over 10 days was measured. Presented are means SEM as percentages of those measured in cultures infected with wild-type NL4-3. uninf, uninfected. 5) or SIVcpzPts (n 7), were slightly more active than HIV-1 M nefs (n 7) (Fig. 5B). However, these differences in viral replication and virus production were not significant (Fig. 5C). Accordingly, similarly to Nef-mediated infectivity enhancement (Fig. 3C), the ability of Nef to stimulate virus replication in human cells obviously did not change much after crossspecies transmission of the virus from chimpanzees to humans. In the next set of experiments we examined the functional activity of nef alleles from HIV-2 and different lineages of SIV. The results demonstrated that nef alleles from SIVgsn, SIVmus, SIVmon, and SIVrcm stimulated HIV-1 replication in human PBMCs about as efficiently as the control HIV-1 NL4-3 nef (Fig. 6A, left panel). In comparison, all six HIV-2 and SIVsmm nef genes analyzed were less active, and NL4-3 recombinants expressing HIV-2 Nefs replicated only slightly more efficiently than the nef-defective control virus (Fig. 6B, middle panel). More nef alleles need to be analyzed to draw definitive conclusions. Nonetheless, in agreement with the results of our previous study using SIVmac239-based constructs (48), the data of the present study suggest that nef alleles from the HIV-2/SIVsmm lineage are usually less active in stimulating viral replication than those from the HIV-1/SIVcpz lineage. In comparison, most Nef proteins from several different, highly divergent primate lentiviruses, i.e., SIVdeb, SIVsyk, SIVsun, SIVtan, and SIVsab, efficiently promoted viral replication in human PBMCs (Fig. 6A, right panel). On average, the three group 1 SIVgsn/mus/mon Nefs that do not downregulate TCR-CD3 enhanced virus production in PBMC cultures slightly more efficiently than the 14 group 2 Nefs ( [n 9] versus [n 42]; P 0.008; all nef alleles were measured in duplicate using two independently generated virus stocks) (Fig. 6C). Our results show that most primate on August 17, 2018 by guest

8 VOL. 81, 2007 Nef-MEDIATED ENHANCEMENT OF VIRAL INFECTIVITY FIG. 6. Replication of X4-tropic HIV-1 recombinants expressing nef alleles from HIV-2 and other heterologous primate lentiviruses. (A) Representative replication kinetics of the indicated HIV-1 nef recombinants. Infections were performed, and virus production was detected as described in the legend to Fig. 3. (B) Average levels of replication of HIV-1 NL4-3 recombinants expressing HIV-2, SIVsmm, SIV-CP, and all other (AO) SIV nef alleles. Results represent mean values of duplicate infections performed with two independent virus stocks of cells derived from one PBMC donor. (C) Cumulative virus production in PBMC cultures inoculated with the indicated NL4-3 nef recombinants relative to wild-type virus. Presented are means SEM. PSL, photon-stimulated luminescence; uninf, uninfected. lentiviral nef alleles promote viral replication in human PBMCs. Unexpectedly, nef alleles from human-derived viruses are usually not more active in promoting viral replication in human cells than those derived from chimpanzees or from different monkey species. Next, we investigated whether the ability of HIV and SIV Nefs to stimulate viral replication may depend on the HIV-1 coreceptor tropism. As shown in Fig. 7A, the potency of the 15 HIV and SIV Nefs in stimulating R5 HIV-1 replication in human PBMCs varied considerably. In agreement with the results obtained using the X4-tropic NL4-3 constructs, some nef genes from SIVcpz, SIVsmm, and several highly divergent SIVs enhanced R5 HIV-1 replication at least as efficiently as those from HIV-1 and HIV-2. To assess in more detail whether the requirement for Nef in HIV-1 replication depends on viral coreceptor usage, we infected PBMCs from a single blood donor with two different virus stocks (each in triplicate) of the X4 and R5 HIV-1 nef recombinants. In agreement with the results of our previous study showing that Nef is required for efficient replication of both X4- and R5-tropic HIV-1 in PBMCs (51), we found that all nef alleles enhanced virus production independently of the viral coreceptor tropism (Fig. 7B). The various HIV and SIV Nef alleles enhanced X4 virus production by 1.7- to 7.8-fold (average standard deviation [SD], 5.4-fold 2.0-fold; n 15) and that of R5 HIV-1 by 3.6- to 23.1-fold (average SD, 11.5-fold 5.1-fold; n 15) compared to the respective nef mutant HIV-1 constructs. This result is in contrast with that of Lundquist and coworkers (38), who reported that Nef is not required for efficient replication of R5-tropic HIV-1 strains in primary CD4 T cells. This apparent contradiction might be due to differences in the culture systems and activation protocols. We observed a significant correlation (R ; P ) between the efficiency of replication of X4- and R5-tropic HIV-1 nef variants (Fig. 7C), suggesting that the viral coreceptor tropism has no major impact on the requirement for Nef in HIV-1 replication in PBMC cultures. Primate lentiviral Nefs enhance HIV-1 replication in HLT ex vivo. It has been shown that the HIV-1 Nef protein promotes viral replication and hence cytopathicity in ex vivo infected HLT (23, 24). This experimental system is most likely highly relevant for virus spread in vivo and the pathogenesis of

9 13860 MÜNCH ET AL. J. VIROL. Downloaded from FIG. 7. Replication of R5-tropic HIV-1 nef recombinants in PBMCs. (A) Representative replication kinetics of the indicated R5-tropic HIV-1 nef recombinants. (B) Cumulative virus production in PBMC cultures inoculated with the indicated X4- or R5-tropic HIV-1 NL4-3 nef recombinants relative to the respective construct expressing the wild-type NL4-3 nef allele. PBMCs from a single blood donor were infected in triplicate (each) with two independent virus stocks. Infections, sampling, and RT assay were all performed in the same experiment. Shown are average values SD of cumulative RT activities measured at days 5, 7, 10, 12, and 14 postinfection. (C) Correlation between the average RT production (n 6) by PBMCs infected with X4- or R5-tropic HIV-1 nef recombinants. Ptt, P. t. troglodytes; uninf, uninfected. PSL, photon-stimulated luminescence. AIDS because it maintains the mixed cell population and complex cytoarchitecture characteristic of HLT (24), a major site of viral replication in HIV-1-infected individuals (20). Furthermore, ex vivo HLT supports productive HIV-1 infection in the absence of exogenous stimulation (24, 25). Therefore, it permits the detection of effects of Nef on T-cell activation that may be missed in other experimental systems. As expected from published data (24, 25, 59), wild-type HIV-1 NL4-3 replicated to higher levels, produced more virus, and caused substantially more CD4 T-cell depletion than the nef-defective control virus (Fig. 8). We found that the seven HIV-1 M, N, and O nef genes as well as the four SIVcpzPtt and SIVcpzPts nef alleles tested all showed similar potencies in enhancing viral replication in ex vivo infected HLT, although the replication kinetics of the HIV-1 constructs expressing the YBF116, ch-nok5, and TAN1 Nefs were slightly delayed (Fig. 8A). On average, the levels of p24 production did not differ significantly between HIV-1 recombinants expressing nef alleles derived from the three groups of HIV-1 and SIVcpzPtt or SIVcpzPts (Fig. 8B). As expected from their efficient replication, all these HIV-1 nef recombinants caused similarly high levels of CD4 T-cell depletion (Fig. 8C). Finally, we investigated the activity of nef alleles from HIV-2, its simian counterpart SIVsmm, and some highly divergent primate lentiviruses, i.e., SIVmon, SIVrcm, SIVdeb, and SIVblu, in HLT ex vivo. As shown in Fig. 9A, all these nef alleles promoted viral replication in ex vivo infected HLT, although the HIV-2 BEN, SIVmon CML1, SIVblu KE31, and SIVrcm GB1 Nefs were only marginally active. This was unexpected because these nef alleles efficiently modulated various cellular receptors including CD4 (60), enhanced virion infectivity (Fig. 2, 3, and 4), and stimulated viral replication in PBMCs (Fig. 6 and 7). The SIVsmm FYR1 and SIVmac239 Nefs increased viral replication and p24 production in histoculture with moderate efficiency, whereas the SIVdeb CM40 Nef was as effective as the highly potent HIV-1 NL4-3 and NA7 Nef alleles (Fig. 9A and B). We found that all HIV-1 Nef recombinants caused more CD4 -T-cell depletion than the nef-defective control virus (Fig. 9C, nef ). There was a strong correlation (R ; P ) between the quantity of on August 17, 2018 by guest

10 VOL. 81, 2007 Nef-MEDIATED ENHANCEMENT OF VIRAL INFECTIVITY FIG. 8. SIVcpz nef alleles enhance HIV-1 replication and CD4 T-cell depletion in HLT ex vivo. For each of the indicated HIV-1 nef variants, 18 tissue blocks were inoculated with virus stocks containing 0.5 ng of p24 antigen and medium was collected every 3 days. (A) Representative replication kinetics of wild-type NL4-3 and the indicated recombinants. (B and C) Average virus production and depletion of CD4 T-cells in HLTs infected ex vivo with the HIV-1 nef recombinants. Tissues from five donors were infected with the indicated nef variants and cumulative p24 production by the tissue blocks over 15 days (B) or CD4 T-cell depletion at the end of culture (C) was determined as described in the methods section. Shown are means SEM of these values as percentages compared to culture infected with the wild-type. PSL, photon-stimulated luminescence. cumulative p24 production and the level of CD4 T-cell depletion (Fig. 9D). Together, the results obtained with HIV-1 recombinants expressing nef alleles from various primate lentiviruses show that they have the capacity to promote viral replication and to enhance CD4 T-cell depletion in ex vivo infected HLT, albeit frequently less efficiently than HIV-1 Nefs. DISCUSSION In the present study, we demonstrate that, similarly to HIV-1 nef alleles, SIVcpz nef alleles derived from naturally infected chimpanzees and also those from a variety of primate lentiviruses that have not been found in humans enhance viral Downloaded from on August 17, 2018 by guest FIG. 9. Replication and cytopathicity of HIV-1 recombinants expressing nef alleles derived from HIV-2 and different SIVs. (A) Representative replication kinetics of the indicated HIV-1 NL4-3 nef recombinants in ex vivo infected HLT. (B) Average amount of p24 released into the culture medium of the tissue blocks infected with the NL4-3 nef variants over a 15-day period. Values in panels B and C give means SEM of tissues from four donors. (C) CD4 T-cell depletion in tissue blocks infected with HIV-1 constructs expressing various HIV and SIV nef alleles. (D) Correlation between p24 virus production and CD4 T-cell depletion in HIV-1-infected HLT ex vivo.

11 13862 MÜNCH ET AL. J. VIROL. infectivity, replication, and cytopathicity in human cells and in ex vivo infected lymphoid tissues. These results expand those of our previous studies showing that nef alleles from unpassaged SIVcpz or SIVsmm strains downmodulate CD4, CD28, CD3 (SIVsmm only), and MHC-I in human cells (37, 48, 62). Accordingly, nef alleles from the simian counterparts of the two human viruses were capable of performing a variety of functions contributing to immune evasion and virus replication without adaptive changes. Notably, our data suggest that this would also be the case for nef alleles from other primate lentiviruses that have not been found in humans. Given that some SIV Nefs show only 30% amino acid sequence homology to those of HIV-1 and HIV-2, it is surprising that many of them can perform all six Nef functions investigated thus far, i.e., downmodulate CD4, CD3, CD28, and MHC-I and promote virion infectivity and stimulate viral replication in human cells or tissues. In fact, we found that some SIV nef alleles are more active in performing specific functions in human cells than all of about 50 HIV-1 and HIV-2 nef genes we have analyzed thus far. However, some SIV nef alleles, i.e., those of SIVmon, SIVrcm, and SIVblu, were poorly active in promoting viral replication and increasing cytopathicity in ex vivo HLT, although they efficiently modulated human cellular receptors, enhanced virion infectivity, and stimulated viral replication in PBMC cultures. It will be of interest to elucidate which changes are required to render these SIV nef alleles fully functional in human tissue. Moreover, previous studies on HIV-1, HIV-2, and SIVmac Nefs showed that some conserved functions are mediated by different domains, suggesting that they may involve different mechanisms (8, 60, 69). Thus, further studies are required to assess which primate lentiviral Nef functions involve conserved molecular mechanisms and which may have evolved independently during primate lentiviral evolution. It is well established that HIV-2 causes lower levels of immune activation and T-cell apoptosis and is usually less pathogenic than HIV-1 (40, 42, 43, 44, 51, 52, 77). One possible reason for the different virulence in the same human host is that HIV-2 is frequently capable of downmodulating TCR- CD3 to suppress T-cell activation and programmed cell death, whereas HIV-1 is unable to perform this protective function (48, 62). Accumulating knowledge suggests, however, that HIV-1 and HIV-2 also differ in other aspects of Nef function. For example, the frequency of defective and/or nonfunctional nef genes seems to be higher in HIV-2 than in HIV-1 infections (71). Lack of Nef function would presumably be associated with low viral loads and, hence, an attenuated clinical course of infection (16, 35, 36). More nef alleles need to be analyzed to make definitive conclusions, but the preliminary data clearly suggest that HIV-2 nef alleles are usually less active than those of HIV-1 in enhancing viral infectivity and replication but that they are equally effective in downmodulating MHC-I and more potent in downregulating the CD28 costimulatory factor of T-cell activation (48). Accordingly, as a group HIV-2 Nefs are highly active in functions facilitating viral immune evasion (i.e., MHC-I down-modulation) and suppressing T-cell activation and apoptosis (i.e., downmodulation of TCR-CD3 and CD28) but not in activities that enhance viral spread in a more direct manner (i.e., enhancement of virion infectivity and stimulation of virus replication). Thus, while the ability to down-modulate TCR-CD3 likely plays a prominent role in the relatively low levels of immune activation observed in HIV-2-infected individuals, a combination of differences in Nef function (and possibly other, as yet unknown viral factors) likely contributes to the low virulence of HIV-2 compared to HIV-1. We have recently performed comprehensive analyses of the ability of HIV-1 and SIVcpz Nefs to modulate various cellular receptors and to alter T-cell activation and programmed death (37, 62). The only definite functional differences were that, on average, SIVcpz Nefs were about twofold more active than HIV-1 Nef in modulating CD28 and the MHC-II-associated invariant chain Ii but less active in downmodulating mature MHC-II (37). In the present study, we show that HIV-1 and SIVcpz nef alleles do not differ significantly in their ability to enhance virion infectivity and to promote viral replication and CD4 T-cell depletion in HLT ex vivo. Further studies are required to elucidate the possible impact of the subtle functional differences between HIV-1 and SIVcpz Nef proteins on T-cell activation and MHC antigen presentation. However, while the possibility that viral properties also play a role cannot be entirely dismissed, our current knowledge suggests that host properties may be more important for the differential clinical outcome of HIV-1 and SIVcpz infection. For instance, a recent study suggests that chimpanzees are less susceptible to disease because their T cells express high levels of sialic acid-binding immunoglobulin-like lectins and are, therefore, less responsive to activation (13, 50). Thus, both the ability of the virus to alter T-cell activation and apoptosis by several mechanisms and the inherent properties of the host contribute to the differential clinical outcome of primate lentiviral infections. One goal of our studies was also to assess whether differences in Nef function may play a role in the differential spread of HIV-1 M, N, and O in the human population. Potentially, lack of Nef function could play a relevant role because this accessory protein enables the virus to persist efficiently in the infected host, and the rate of sexual transmission accounting for most HIV-1 infections correlates with the viral load (27, 58). We have previously shown that nef alleles from the three groups of HIV-1 modulate CD4, CD28, MHC-I, MHC-II, and Ii surface expression with comparable efficiency (37, 62, 63). Here, we demonstrate that group M, N, and O Nefs also enhance virion infectivity and viral replication and cytopathicity in PBMCs and lymphoid tissue ex vivo with similar efficiencies. Thus, differences in Nef function do not explain the different distribution of the HIV-1 M, N, and O groups. The result that HIV-1 M, O, and N do not show definite differences in Nef function is in agreement with published data showing that these three groups show similar levels of pathogenicity (4, 18, 67). We also found that SIVcpz nef alleles from the P. t. troglodytes and P. t. schweinfurthii subspecies of chimpanzee are both equally active as HIV-1 nef alleles in human cells and lymphoid tissues. Thus, subspecies-specific differences in Nef function do not explain why only SIVcpzPtt has been found in humans. In sum, our analysis of nef alleles from a large variety of primate lentiviruses shows that most of them are capable not only of modulating the surface expression of various receptor but also of enhancing viral infectivity and replication in human cells. Altogether, the data also reveal that despite their ability to perform a striking variety of activities HIV-1 Nefs are