Macrophage Derived SIV Exhibits Enhanced. Infectivity by Comparison with T Cell Derived Virus ACCEPTED

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1 JVI Accepts, published online ahead of print on November 00 J. Virol. doi:./jvi.01-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Macrophage Derived SIV Exhibits Enhanced Infectivity by Comparison with T Cell Derived Virus Peter J. Gaskill 1, Michelle Zandonatti 1, Tim Gilmartin, Steven R. Head and Howard S. Fox 1 1 Molecular and Integrative Neurosciences Department; DNA Microarray Core Facility The Scripps Research Institute, 0 N. Torrey Pines Road, La Jolla, CA 0 Running title: Enhanced infectivity of macrophage derived SIV 1 Table, Figures Word count: Abstract: words, Text:,00 words Correspondence should be addressed to: Dr. Howard S. Fox The Scripps Research Institute 0 North Torrey Pines Road SP0-00 La Jolla, California, 0 USA Phone: () -; Fax: () - hsfox@scripps.edu 1

2 ABSTRACT HIV and SIV infect and productively replicate in macrophages and T lymphocytes. Here, we show that SIV virions derived from macrophages have higher levels of infectivity compared with those derived from T cells. The lower infectivity of T cell derived viruses is influenced by the quantity or type of mannose residues on the virion. Our results demonstrate that the cellular origin of a virus is a major factor in viral infectivity. Cell-type specific factors in viral infectivity, and of organ or disease-stage specific differences in cellular derivation of virions, can be critical in the pathogenesis of HIV and AIDS.

3 CD+ T cells and macrophages are the major targets and sources of HIV and SIV. In view of the essential but highly distinct roles these cell types play in the pathogenesis of HIV (,, 1, ), understanding how viral production in each of these cell types affects the subsequent activity of virions is important to understanding the development of disease. To determine if different factors to which virions are exposed during their generation can influence their infectivity, we generated SIV stocks from both macrophages and T cells. Two different molecular clones of SIV, SIVmac1 (1) and SIVmac1 (), were used to generate viral stocks from both macrophages and T cells. These SIV stocks (hereafter referred to as matching viral stocks) were derived from infection of primary rhesus monocyte-derived macrophages (MDM) or primary rhesus T cells. Primary MDM were derived from freshly isolated rhesus PBMC by immunomagnetic CDb selection and differentiated by adherence for days in the presence of M-CSF ( ng/ml). The remaining PBMC were then subjected to CD immunomagnetic depletion to yield CD+ enriched cells for the primary T cell cultures, and then stimulated with PHA ( µg/ml) and IL- ( ng/ml) for days. Following stimulation, T cells were cultured in the presence of IL- alone for additional days. Both MDM and T cells were inoculated with SIV after six days in culture and cellfree supernatants from each cell type were collected daily for eight days after SIV inoculation and stored at -0 C. Stocks were then pooled and either aliquoted and frozen, or purified by ultracentrifugation over a 0% sucrose cushion to eliminate contaminating cellular-derived factors such as cytokines, then aliquoted and stored at -0 C. Stocks from macrophages and T-cells were collected, stored and processed simultaneously to avoid

4 preparation related differences in infectivity. Viral stocks were generated from a total of twelve different rhesus donors; only macrophage and T-cell stocks derived from cells isolated from a single donor at the same time were used for comparison. SIV stocks were quantified by both ELISA for p Gag (Beckmann-Coulter) and bdna assay (Bayer Reference Testing Laboratory) for viral genome quantification. These assays showed no significant difference between the matching viral stocks in terms of copies of viral RNA per pg of p Gag. Further testing by immunoblot (using the KK antibody to detect Env, and FA to detect Gag) indicated that the ratio of Env to Gag was constant in matching viral stocks. This is consistent previous studies that virions produced in T cells and macrophages have similar levels of the Env (, ), indicating that the virus encoded aspects of the virion are likely similar in macrophage and T cell derived virions. Initial experiments used equivalent amounts of non-ultracentrifuge purified SIVmac1 matching viral stocks, derived from primary cultures from three different monkeys, to infect primary rhesus MDM from two different donors. In each case, to - fold higher levels of infection was achieved by the macrophage-derived versus the T-cell derived stocks, determined by both number of cells infected (by immunofluorescence) and the level of p (by ELISA) in the supernatant (data not shown). Since both macrophages and T cells make cytokines, growth factors and other molecules in response to infection, ultracentrifuge purified viral stocks were then used to remove potential contribution of these cellular products that could alter virion infectivity from viral stocks. Equivalent amounts of purified SIVmac1 matching viral stocks were then used to infect primary rhesus MDM derived from two different donors. Inoculation with SIV derived from macrophages again resulted in higher levels of infectivity than did inoculation with SIV

5 derived from T cells (n=) (Figure 1A-C). The greater number of cells infected by macrophage-derived virus corresponds with the increase in p production, indicating that macrophage-derived virions are able to infect cells more effectively than T-cell derived virions. As in this experiment, subsequent studies performed with both unpurified and purified viral stocks yielded similar results; the data reported below utilized the purified stocks. Additional studies were performed via infection of a number of different cell types with matching viral stocks derived from the different SIV strains. Cell types infected included two indicator lines, CCR expressing (Hi-) GHOST cells (HOS derivative, which produce GFP in response to infection), LuSIV cells (1xCEM derivative, which produce luciferase in response to infection), as well as primary rhesus MDM and primary rhesus T- cells to better examine the potential in vivo relevance of this effect. Infection of primary cells was performed in MDM derived from six different macaques and in T-cells derived from three different macaques. These infections were performed via spinoculation ( hours at 1,00 x g at C), to minimize the effects of virion diffusion and attachment on infection and to synchronize the infections by facilitating virion attachment to the cell surface (1, 1). Infections were analyzed by ELISA quantification of supernatant p levels. All infections in both the indicator and primary cells with matching viral stocks (Figure A-E) resulted in a greater level of infection by macrophage-derived viral stocks than by T-cell derived viral stocks. The increased infectivity of macrophage derived virus over T-cell derived virus in multiple cell types using different viral strains demonstrates that the enhanced infectivity of macrophage-derived virions is not dependent on viral strain or

6 on target cell type, and is dependent only on the cell type in which the virions were produced. The enhanced infectivity of macrophage-derived virus could result from stable changes to the virus such as alterations in its genome, or from transient changes such as addition of cellular proteins to the viral surface. We generated two different twice passaged viral stocks of SIVmac1 by generating viral stocks in either macrophages or T cells and subsequently passaging those stocks through the other cell type, i.e. macrophage-derived stocks were secondarily passaged in T cells and T cell derived stocks were secondarily passaged in macrophages. Identical amounts of both types of twice passaged stocks were then used to infect primary MDM derived from two rhesus macaques. The macrophage- derived stocks secondarily passaged in T-cells generated much lower levels of infection than the T cell derived stocks were secondarily passaged in macrophages (Figure F). Thus, the viral stocks functioned like virus derived from the cells in which they were last passaged, rather than the cells from which they were initially derived. Sequence analysis of three independent molecular clones of full length gp derived from viral genomic RNA from each twice passaged stock revealed no differences (data not shown), further supporting the idea that the enhanced infectivity results from transient changes to the virions. To determine if generation in either macrophages or T cells altered virion infectivity through changes in viral attachment capacity, an attachment assay (modified from (0)) was performed using Hi- GHOST cells, revealing no significant difference in attachment for matched stocks derived from two different monkeys (Figure G). Additionally, Hi- GHOST cells were infected with identical amounts of matching SIVmac1 (Figure H)

7 and SIVmac1 (data not shown) stocks by both spinoculation and standard inoculation. In both types of inoculation macrophage derived virions produced higher levels of infection than did those derived from T cells, indicating that the increased infectivity of macrophagederived viral particles is not due to increased or decreased attachment capacity. As cell-type specific variability in the glycosylation of HIV and SIV envelope proteins has been reported (-1) and virion glycosylation has been shown to affect HIV and SIV infectivity (, 1), we examined differences in virion glycosylation pathways as another possible mechanism for the increased infectivity of macrophage derived virus. Microarray analysis was used to examine differences in the expression of genes involved in glycosylation processes between uninfected primary macrophages and T cells from three different donors grown under the conditions used to produce our viral stocks. A large number () of gene transcripts differed significantly (p < 0.01) in expression between macrophages and T-cells. A total of genes, involved in glycan degradation and 1 involved in glycan transfer, were at least -fold higher in macrophages as compared to T- cells and another 1 genes whose products are involved in glycan transferase activity were at least two-fold higher in T cells as compared to macrophages (Table 1). Differential expression of two representative genes was corroborated by quantitative real time PCR analyses, demonstrating increased expression of the mannosidase MANB1 gene in macrophages (1.0 ±.0 fold in the three donors, compared to their respective T-cells) and increased expression of the sialyltransferase STGAL1 gene in T-cells (.1 ± 1. fold). To examine the functional effects of differential glycosylation of viral particles, we removed glycans from virion surfaces using different glycosidases, then examined the infectivity of glycosidase treated matching stocks in LuSIV indicator cells. Infection of these

8 cells with α--,,,-neuraminidase-treated matching stocks of SIVmac1 led to similar, small changes in infectivity in both macrophage and T-cell derived viral stocks (Figures A, B). In contrast, treatment of matching stocks with α-1-,-mannosidase showed no significant enhancement in infectivity of macrophage derived virions (Figure C), but a three and a half-fold increase in infectivity of T-cell derived virions (p < 0.01, n=, ANOVA with Tukey s post-hoc test) (Figure D). Additional infections were performed with matching viral stocks treated with a distinct mannosidase enzyme, α-1-,,-mannosidase, to examine more complete removal of mannose from the virion. Macrophage derived stocks of SIVmac1 treated with α-1-,,-mannosidase demonstrated a significant, but slight increase in infectivity of less than -fold (Figure E, p<0.01, n=, ANOVA with Tukey s post-hoc test). As with α-1-,-mannosidase treatment, T-cell derived stocks treated with α-1-,,-mannosidase demonstrated a large increase in infectivity of up to nine-fold (Figure F, p<0.01, n=, ANOVA with Tukey s post-hoc test). Changes in gp and gp1 glycosylation patterns can strongly alter the infectivity of different strains of HIV (-1). Several studies show that desialylation of viral particles via neuraminidase digestion enhanced the HIV/SIV infectivity (, 1, 1, 1, 1), in general agreement with the findings in this report. The discovery that mannosidase treatment increases the infectivity of virions derived from T-cells differs from previous reports (, 1). However, the cellular origin of a virion is crucial to examination of glycosylation mediated effects on infectivity due to differential exposure to a variety of different glycosylation related pathways and enzymes during virion production (,, ) and unlike in previous studies the virus in this report was generated in primary macrophages and T-cells.

9 The data discussed above demonstrate a significant cell-type specific variation in the infectivity of macrophage and T cell derived SIV particles, and suggest that the greater infectivity of macrophage derived virions over T cells derived virions is due to a lower number of mannose residues on the surface of viral particles derived from macrophages. Support for this idea comes from data revealing that the glycosylation patterns of the heavily glycosylated, mannose rich gp (, ), differ in a host cell specific fashion (, -1). We also find that several genes whose products degrade glycans are increased in macrophages. The fact that the majority of the glycoconjugates on gp contain high mannose glycans (, ) further strengthens the possibility that mannose residues on gp affect infectivity. On the other hand, as opposed to effects on viral proteins, differential glycosylation of numerous cellular proteins incorporated into virions in a cell type dependent manner (1) could also change viral infectivity. These data show that the viral infectivity of SIV is influenced by the cellular origin of the virion, and that macrophages generate virions with greater levels of infectivity than those generated by T cells. The data suggest that these differences may be due to differences in the number or arrangement of mannose residues on the surface of macrophage and T cell derived virions. The implications of this finding are important in the context of HIV infection and disease progression, particularly in the study of transmission, the dynamics of viral spread, and organ-specific viral evolution and pathogenesis. As macrophages and T cells each play distinct roles in disease, the finding that virus derived from one or the other cell type may behave differently has important implications for our understanding of HIV pathogenesis.

10 We thank the members of the Fox lab for their many contributions, Dr. Michael Buchmeier for his many helpful suggestions, Dr. Phillipe Gallay for advice on the attachment assay, Dr. J. Lindsay Whitton for use of fluorescence microscopy equipment, and the NIH AIDS and Reference Reagent Program for cell lines and monoclonal antibodies. This work was supported by NIH grants MH01, MH00, and NS0; the Gene Microarray Core resources and collaborative efforts provided by The Consortium for Functional Glycomics funded by GM; PJG was supported by T AI00. This is manuscript #1 from The Scripps Research Institute.

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14 Accession Gene Probe set Gene Name Number Symbol Glycan Degradation Increased in Macrophages MΦ T cell Fold 0_at NM_000. ARSA ARYLSULFATASE A _at NM_ ARSB ARYLSULFATASE B NM_001_s_at NM_001 ASAH1 N-ACYLSPHINGOSINE AMIDOHYDROLASE (ACID CERAMIDASE) _at NM_ FUCA1 FUCOSIDASE, ALPHA-L- 1, TISSUE _at NM_0001. GAA GLUCOSIDASE, ALPHA; ACID _at NM_ GALC GALACTOSYLCERAMIDASE _at NM_0001. GALNS GALACTOSAMINE (N-ACETYL)--SULFATE SULFATASE _s_at K00.1 GLUCOSIDASE, BETA; ACID (INCLUDES GBA 00_at K00.1 GLUCOSYLCERAMIDASE) 0... _at NM_ GLA GALACTOSIDASE, ALPHA _at NM_ GLB1 GALACTOSIDASE, BETA _at M.1 GMA GM GANGLIOSIDE ACTIVATOR _at NM_000.1 GNS GLUCOSAMINE (N-ACETYL)--SULFATASE... 00_at NM_000.1 GUSB GLUCURONIDASE, BETA _at AL1 HEXA HEXOSAMINIDASE A (ALPHA POLYPEPTIDE) _at NM_0001. HEXB HEXOSAMINIDASE B (BETA POLYPEPTIDE) _at U.1 MANB1 MANNOSIDASE, ALPHA, CLASS B, MEMBER _at NM_000.1 MANBA MANNOSIDASE, BETA A, LYSOSOMAL _at M0.1 NAGA N-ACETYLGALACTOSAMINIDASE, ALPHA _s_at NM_000.1 NAGLU N-ACETYLGLUCOSAMINIDASE, ALPHA-...0 BC000_x_at BC NEU1 SIALIDASE 1 (LYSOSOMAL SIALIDASE) 0_s_at U N-ACETYLNEURAMINATE PYRUVATE AF_at AF NPL LYASE PROTECTIVE PROTEIN FOR BETA- 001_at NM_ PPGB 0... GALACTOSIDASE STEROID SULFATASE (MICROSOMAL), 0_at AI1 STS 1... ARYLSULFATASE C, ISOZYME S _s_at NM_ SULF SULFATASE _at NM_

15 Glycan Transferase Increased in Macrophages AB0_s_at AB0 BGNT1 1_at NM_00 BGNT 1_x_at NM_1 BGNT 1_at NM_00. BGALT _at NM_1 CHST1 0_at NM_01.1 CHST UDP-GLCNAC:BETAGAL BETA-1,-N- ACETYLGLUCOSAMINYLTRANSFERASE 1 UDP-GLCNAC:BETAGAL BETA-1,-N- ACETYLGLUCOSAMINYLTRANSFERASE UDP-GLCNAC:BETAGAL BETA-1,-N- ACETYLGLUCOSAMINYLTRANSFERASE UDP-GAL:BETAGLCNAC BETA 1,- GALACTOSYLTRANSFERASE, POLYPEPTIDE CARBOHYDRATE (CHONDROITIN ) SULFOTRANSFERASE 1 ACETYLGLUCOSAMINE -O) SULFOTRANSFERASE CARBOHYDRATE (N- UDP-N-ACETYL-ALPHA-D- 1_x_at NM_ GALACTOSAMINE:POLYPEPTIDE N- GALNT ACETYLGALACTOSAMINYLTRANSFERASE 1_at NM_ _at NM_01. GBGT1 0_s_at NM_1 GCNT GLOBOSIDE ALPHA-1,-N- ACETYLGALACTOSAMINYLTRANSFERASE 1 GLUCOSAMINYL (N-ACETYL) TRANSFERASE, I-BRANCHING ENZYME _at NM_01 GLCE GLUCURONYL C-EPIMERASE _s_at NM_00.1 HSST1 SULFOTRANSFERASE _s_at NM_00 HEPARAN SULFATE (GLUCOSAMINE) -O-... 1_at 0_s_at NM_000.1 HSST NM_000. MGAT1 SULFOTRANSFERASE MANNOSYL (ALPHA-1,-)-GLYCOPROTEIN ACETYLGLUCOSAMINYLTRANSFERASE HEPARAN SULFATE (GLUCOSAMINE) -O- BETA-1,-N- MANNOSYL (ALPHA-1,-)-GLYCOPROTEIN _at NM_ BETA-1,-N- MGATB 01_s_at NM_01.1 ACETYLGLUCOSAMINYLTRANSFERASE, _s_at 00_at NM_001 NM_001. NDST1 ISOZYME B N-DEACETYLASE/N-SULFOTRANSFERASE (HEPARAN GLUCOSAMINYL)

16 _s_at 0_at 1_at Increased in T cells NM_000. STGAL NM_00.1 STGALNAC NM_00. UGCGL 1_at NM_01 ALG ST BETA-GALACTOSIDE ALPHA-,- SIALYLTRANSFERASE ST (ALPHA-N-ACETYL-NEURAMINYL-,- BETA-GALACTOSYL-1,)-N- ACETYLGALACTOSAMINIDE ALPHA-,- SIALYLTRANSFERASE UDP-GLUCOSE CERAMIDE GLUCOSYLTRANSFERASE-LIKE ASPARAGINE-LINKED GLYCOSYLATION GLUCOSYLTRANSFERASE) HOMOLOG (YEAST, ALPHA-1,- 1_s_at NM_001. BETA-1,-N _s_at NM_01 BGALNT1 ACETYLGALACTOSAMINYLTRANSFERASE. 1.. _at 1_at NM_01 NM_001.1 C1GALT1 _at NM_0 CHGN 01_at NM_00.1 CHST 00_at NM_0 CHSY CORE 1 SYNTHASE, GLYCOPROTEIN-N- ACETYLGALACTOSAMINE -BETA- GALACTOSYLTRANSFERASE, 1 CHONDROITIN BETA1, N- ACETYLGALACTOSAMINYLTRANSFERASE CARBOHYDRATE (N- ACETYLGLUCOSAMINE--O) SULFOTRANSFERASE CARBOHYDRATE (CHONDROITIN) SYNTHASE _at NM_ EXT1 EXOSTOSES (MULTIPLE) _at NM_00. FUT FUCOSYLTRANSFERASE (ALPHA (1,) FUCOSYLTRANSFERASE)...1 0_s_at NM_000.1 FUT FUCOSYLTRANSFERASE).1.0. _s_at NM_ FUCOSYLTRANSFERASE (ALPHA (1,)... 01_s_at NM_ _s_at NM_000.1 MFNG MANIC FRINGE HOMOLOG (DROSOPHILA) _s_at NM_ OGT 01_at NM_.1 STGAL1 O-LINKED N-ACETYLGLUCOSAMINE (GLCNAC) TRANSFERASE (UDP-N- ACETYLGLUCOSAMINE:POLYPEPTIDE-N- ACETYLGLUCOSAMINYL TRANSFERASE) ST BETA-GALACTOSAMIDE ALPHA-,- SIALYLTRANFERASE

17 Table 1 Probe sets identifying genes involved in glycan degredation and glycan transferase that show significant changes between macrophages (MΦ) and T cells. Numerical values for each cell type indicate the geometric mean intensity from the three independent samples. 1

18 FIGURE LEGENDS Figure 1 Macrophage derived virus has greater infectivity than T-cell derived virus. Monocyte derived macrophages plated on chamber slides were inoculated in triplicate with SIVmac1 derived from macrophages and T-cells. Slides were stained to determine percentage of cells infected and supernatants were analyzed for p levels. (A,B) Examples of immunofluorescence (Red anti-gag visualized with rhodamine, Blue cell nuclei visualized with DAPI) of cells infected with matched stocks of macrophage derived virions (A) and T-cell derived virions (B). (C) Percentage of macrophages infected with macrophage and T-cell derived virus (inoculated using the p Gag levels indicated) is designated by the bars, and virus in the supernatant (measured by p Gag) is indicated by the symbols and lines. The percentage of macrophages infected was determined by counting all the cells on a slide, thus there is no error given for this measurement. Figure Cellular origin determines infectivity independently of viral strain and target cell. Hi- GHOST cells were spinoculated in triplicate with different concentrations of SIVmac 1 (A, B) or SIVmac1 (B) derived from either macrophages or T-cells, then fixed at hours and analyzed by flow-cytometry for GFP expression to determine the percentage of cells infected. (C) LuSIV cells were spinoculated in sextuplet with SIVmac1 derived from primary macrophages or T-cells, then analyzed by luminescence 0 hours post infection to determine the level of infectivity. (D) Primary macrophages were spinoculated in triplicate with the indicated amounts of SIVmac1 generated in primary T-cells and primary macrophages, then supernatants were analyzed for days post-infection for p 1

19 production. (E) Primary CD+ T-cells were infected with SIVmac1 derived from primary T-cells and macrophages, then supernatants were analyzed for days post-infection for p production. (F) Doubly-passaged infection. SIVmac1 generated in primary macrophages or T cells and then passaged through the other cell type, either macrophages or T-cells, was used for infection of macrophages by spinoculation. Supernatants were collected for days and analyzed for p production. (G) Matched SIVmac1 virus stocks, produced in cells derived from rhesus macaques 1 or 1, were added, at C, to Hi- GHOST cells for 0 minutes, washed, and total RNA isolated. Relative amounts of SIV adhered to the cells was determined by real-time quantitative PCR using primers for SIV and 1S RNA using the dct method. (H) Standard inoculation versus spinoculation. SIVmac1 generated in primary T-cells and macrophages was used to infect Hi- GHOST cells by standard inoculation and spinoculation. Cells were analyzed at hours by FACS. Figure Removal of mannose from virion surface affects infectivity. SIVmac1 derived from primary macrophages (A, C, E) or T-cells (B, D, F) was digested with the indicated concentrations of glycosidases; and used to infect LuSIV cells by spinoculation. Cells were then analyzed by luminescence 0 hours post infection. All statistical analyses performed by ANOVA with Tukey s post-hoc test, n= for each experiment. 1

20 CCEPTE

21 CCEPTED

22