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Journal of Virology, February 2008, p. 1615-1621, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01757-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Macrophage-Derived Simian Immunodeficiency Virus Exhibits Enhanced Infectivity by Comparison with T-Cell-Derived Virus

Peter J. Gaskill,¹ Michelle Zandonatti,¹ Tim Gilmartin,² Steven R. Head,² and Howard S. Fox^1*

Molecular and Integrative Neurosciences Department,¹ DNA Microarray Core Facility, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037²

Received 10 August 2007/ Accepted 13 November 2007

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Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infect and productively replicate in macrophages and T lymphocytes. Here, we show that SIV virions derived from macrophages have higher levels of infectivity than 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 organ-specific or disease stage-specific differences in cellular derivation of virions, can be critical in the pathogenesis of HIV and AIDS.

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CD4⁺ T cells and macrophages are the major targets and sources of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). In view of the essential but highly distinct roles these cell types play in the pathogenesis of HIV (7, 9, 14, 22), understanding how viral production in each of these cell types affects the subsequent activity of virions is important for 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 molecular clones of SIV, SIVmac316 (17) and SIVmac129 (6), were used to generate viral stocks from both macrophages and T cells. These SIV stocks (herein 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 peripheral blood mononuclear cells by immunomagnetic CD11b selection and differentiated by adherence for 6 days in the presence of macrophage colony-stimulating factor (10 ng/ml). The remaining peripheral blood mononuclear cells were then subjected to CD8 immunomagnetic depletion to yield CD4⁺ enriched cells for the primary T-cell cultures and then stimulated with phytohemagglutinin (5 µg/ml) and interleukin 2 (10 ng/ml) for 3 days. Following stimulation, T cells were cultured in the presence of interleukin 2 alone for 3 additional days.

Both MDM and T cells were inoculated with SIV after 6 days in culture, and cell-free supernatants from each cell type were collected daily for 8 days after SIV inoculation and stored at –80°C. Stocks were then pooled and either aliquoted and frozen or purified by ultracentrifugation over a 20% sucrose cushion to eliminate contaminating cell-derived factors such as cytokines and then aliquoted and stored at –80°C. Stocks from macrophages and T cells were collected, stored, and processed simultaneously to avoid preparation related differences in infectivity. Viral stocks were generated from a total of 12 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 enzyme-linked immunosorbent assay (ELISA) for p27 Gag (Beckman-Coulter) and branched-DNA 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 p27 Gag. Further testing by immunoblotting (using the KK45 antibody to detect Env and FA2 to detect Gag) indicated that the ratio of Env to Gag was constant in matching viral stocks. This is consistent with previous studies showing that virions produced in T cells and macrophages have similar levels of Env (2, 3), 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 SIVmac316 matching viral stocks, derived from primary cultures from three monkeys, to infect primary rhesus MDM from two donors. In each case, 3- to 10-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 p27 (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 any potential contribution of these cellular products that could alter virion infectivity from viral stocks. Equivalent amounts of purified SIVmac316 matching viral stocks were then used to infect primary rhesus MDM derived from two donors. Inoculation with SIV derived from macrophages again resulted in higher levels of infectivity than did inoculation with SIV derived from T cells (n = 4) (Fig. 1). The greater number of cells infected by macrophage-derived virus corresponds with the increase in p27 production, indicating that macrophage-derived virions are able to infect cells more effectively than T-cell-derived virions. Subsequent studies performed with both unpurified and purified viral stocks yielded similar results; the data reported below were obtained with the purified stocks.

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FIG. 1. Macrophage-derived virus has greater infectivity than T-cell-derived virus. Monocyte derived macrophages plated on chamber slides were inoculated in triplicate with SIVmac316 derived from macrophages and T cells. Slides were stained to determine percentages of cells infected, and supernatants were analyzed for p27 levels. (A and B) Examples of immunofluorescence (red, anti-Gag visualized with rhodamine; blue, cell nuclei visualized with DAPI [4',6'-diamidino-2-phenylindole]) of cells infected with matched stocks of macrophage-derived virions (A) and T-cell-derived virions (B). (C) Bars show percentages of macrophages infected with macrophage and T-cell-derived virus (inoculated using the p27 Gag levels indicated); symbols and lines show levels of virus in supernatants (measured by p27 Gag). The percentage of macrophages infected was determined by counting all the cells on a slide; thus, no error is given.

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, CCR5-expressing (Hi-5) GHOST cells (HOS derivative, which produce green fluorescent protein in response to infection), LuSIV cells (174xCEM derivative, which produce luciferase in response to infection), and 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 macaques and in T cells derived from three macaques. These infections were performed via spinoculation (2 h at 1,200 x g at 25°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 (18, 19). Infections were analyzed by ELISA quantification of supernatant p27 levels.

All infections in both the indicator and primary cells with matching viral stocks (Fig. 2A to 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 on target cell type but rather is dependent only on the cell type in which the virions were produced.

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FIG. 2. Cellular origin determines infectivity independently of viral strain and target cell. Hi-5 GHOST cells were spinoculated in triplicate with different concentrations of SIVmac 316 (A, B) or SIVmac129 (B) derived from either macrophages or T cells, fixed at 48 h, and analyzed by flow cytometry for green fluorescent protein expression to determine the percentage of cells infected. (C) LuSIV cells were spinoculated (six replicates) with SIVmac316 derived from primary macrophages or T cells and then analyzed by luminescence 40 h postinfection to determine the level of infectivity. (D) Primary macrophages were spinoculated in triplicate with the indicated amounts of SIVmac316 generated in primary T cells and primary macrophages, and then supernatants were analyzed for 6 days postinfection (P.I.) for p27 production. (E) Primary CD4+ T cells were infected with SIVmac316 derived from primary T cells and macrophages, and then supernatants were analyzed for 6 days postinfection for p27 production. (F) Doubly passaged infection. SIVmac316 generated in primary macrophages or T cells and then passaged through the other cell type was used for infection of macrophages by spinoculation. Supernatants were collected for 6 days and analyzed for p27 production. (G) Matched SIVmac316 virus stocks, produced in cells derived from two rhesus macaques (516 and 521), were added at 4°C to Hi-5 GHOST cells for 30 min, and then cells were washed and total RNA was isolated. Relative amounts of SIV adhering to the cells were determined by real-time quantitative PCR using primers for SIV and 18S RNA by the method. (H) Standard inoculation versus spinoculation. SIVmac316 generated in primary T cells and macrophages was used to infect Hi-5 GHOST cells by standard inoculation and spinoculation. Cells were analyzed at 48 h by FACS.

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 SIVmac316 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 secondarily passaged in macrophages (Fig. 2F). 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 gp120 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 reference 20) was performed using Hi-5 GHOST cells, revealing no significant difference in attachment for matched stocks derived from two different monkeys (Fig. 2G). Additionally, Hi-5 GHOST cells were infected with identical amounts of matching SIVmac316 (Fig. 2H) and SIVmac129 (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 macrophage-derived 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 (10-12) and virion glycosylation has been shown to affect HIV and SIV infectivity (8, 15), 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 donors grown under the conditions used to produce our viral stocks. A large number (344) of gene transcripts differed significantly (P < 0.01) in expression between macrophages and T cells. A total of 42 genes, 24 involved in glycan degradation and 18 involved in glycan transfer, were at least twofold higher in macrophages than in T cells, and another 12 genes whose products are involved in glycan transferase activity were at least twofold higher in T cells than in macrophages (Table 1). Differential expression of two representative genes was corroborated by quantitative real-time PCR analyses, demonstrating increased expression of the mannosidase MAN2B1 gene in macrophages (15.0-fold ± 3.0-fold in the three donors, compared to their respective T cells) and increased expression of the sialyltransferase ST6GAL1 gene in T cells (6.1-fold ± 1.5-fold, compared to their respective macrophages).

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TABLE 1. Probe sets identifying genes involved in glycan degradation and glycan transferase that show significant changes between macrophages and T cells

To examine the functional effects of differential glycosylation of viral particles, we removed glycans from virion surfaces using different glycosidases and then examined the infectivity of glycosidase-treated matching stocks in LuSIV indicator cells. Infection of these cells with -2,3,6,8,9-neuraminidase-treated matching stocks of SIVmac316 led to similar, small changes in infectivity in both macrophage and T-cell-derived viral stocks (Fig. 3A and B). In contrast, treatment of matching stocks with -1,2,3-mannosidase showed no significant enhancement in infectivity of macrophage-derived virions (Fig. 3C) but a 3.5-fold increase in infectivity of T-cell-derived virions (P < 0.01, n = 6, analysis of variance [ANOVA] with Tukey's post hoc test) (Fig. 3D).

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FIG. 3. Removal of mannose from the virion surface affects infectivity. SIVmac316 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 40 h postinfection. All statistical analyses performed by ANOVA with Tukey's post hoc test; n = 6 for each experiment.

Additional infections were performed with matching viral stocks treated with a distinct mannosidase enzyme, -1,2,3,6-mannosidase, to examine more complete removal of mannose from the virion. Macrophage-derived stocks of SIVmac316 treated with -1,2,3,6-mannosidase demonstrated a significant but slight increase in infectivity of less than twofold (Fig. 3E; P < 0.01, n = 6, ANOVA with Tukey's post hoc test). As with -1,2,3-mannosidase treatment, T-cell-derived stocks treated with -1,2,3,6-mannosidase demonstrated a large increase in infectivity, up to ninefold (Fig. 3F; P < 0.01, n = 6, ANOVA with Tukey's post hoc test).

Changes in gp120 and gp41 glycosylation patterns can strongly alter the infectivity of different strains of HIV (10-12). Several studies show that desialylation of viral particles via neuraminidase digestion enhanced the HIV/SIV infectivity (8, 13, 15, 16, 21), 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 (8, 15). 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 (10, 11, 23), and unlike in previous studies, the virus in this report was generated in primary macrophages and T cells.

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-cell-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 gp120 (4, 24) differ in a host cell-specific fashion (5, 10-12). We also find that several genes whose products degrade glycans are increased in macrophages. The fact that the majority of the glycoconjugates on gp120 contain high-mannose glycans (4, 24) further strengthens the possibility that mannose residues on gp120 affect infectivity. On the other hand, differential glycosylation of numerous cellular proteins incorporated into virions in a cell type-dependent manner (1), as opposed to effects on viral proteins, 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 T cells. The data suggest that these differences may be due to differences in the number or arrangement of mannose residues on the surfaces 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 viruses derived from the different cell types may behave differently has important implications for our understanding of HIV pathogenesis.

 
We thank the members of the Fox lab for their many contributions, Michael Buchmeier for his many helpful suggestions, Phillipe Gallay for advice on the attachment assay, J. Lindsay Whitton for use of fluorescence microscopy equipment, and the NIH AIDS Research and Reference Reagent Program for cell lines and monoclonal antibodies.

This work was supported by NIH grants MH062261, MH073490, and NS045534 and by the Gene Microarray Core resources and collaborative efforts provided by The Consortium for Functional Glycomics funded by GM62116. P.J.G. was supported by T32 AI07606.

This is manuscript 18383 from The Scripps Research Institute.

 
* Corresponding author. Mailing address: The Scripps Research Institute, 10550 North Torrey Pines Road, SP30-2030, La Jolla, CA 92037. Phone: (858) 784-7171. Fax: (858) 784-7296. E-mail: hsfox{at}scripps.edu

Published ahead of print on 28 November 2007.

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Journal of Virology, February 2008, p. 1615-1621, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01757-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gaskill, P. J. Articles by Fox, H. S. Search for Related Content PubMed PubMed Citation Articles by Gaskill, P. J. Articles by Fox, H. S.