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Journal of Virology, April 2005, p. 4944-4951, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4944-4951.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

CD4-Independent Entry and Replication of Simian Immunodeficiency Virus in Primary Rhesus Macaque Astrocytes Are Regulated by the Transmembrane Protein

Emily D. Overholser, Tahar Babas, M. Christine Zink, Sheila A. Barber, and Janice E. Clements*

The Retrovirus Laboratory, Department of Comparative Medicine and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland

Received 5 March 2004/ Accepted 1 December 2004


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ABSTRACT
 
Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the env and nef genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the nef gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.


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INTRODUCTION
 
Human immunodeficiency virus (HIV) enters the central nervous system (CNS) and causes encephalitis in approximately 20 to 30% of HIV-positive individuals (32). Simian immunodeficiency virus (SIV) provides the best animal model to study HIV encephalitis. Twenty to thirty percent of infected macaques develop CNS lesions similar to those noted in HIV-infected individuals in addition to developing AIDS. In the SIV model, perivascular macrophages and microglia (29, 54, 55) are the major target cells associated with viral infection in the brain; however, astrocytes are also susceptible to infection by SIV (22, 37), as they are to infection by HIV (45, 48). Astrocytes are the most numerous cells in the brain and are responsible for a variety of functions, including maintenance of CNS homeostasis, formation of the structural framework for complex neural networks, immune surveillance, antigen presentation, and stimulation of endothelial cells to form tight junctions, thereupon forming the blood-brain barrier (7, 13, 39, 43). Because of their essential functions in the CNS, the susceptibility of astrocytes to lentiviral infection may have important pathogenic consequences for HIV- or SIV-associated CNS disease. For example, human fetal astrocytes infected with HIV type 1 (HIV-1) or exposed to HIV-1 surface glycoprotein (SU) exhibited an impaired ability in glutamate uptake, which was linked to down-regulated expression of EAAT1 and EAAT2, the glutamate transporters (50). Thus, the identification of the genetic determinants of HIV or SIV required for infection of astrocytes is crucial for the development of new strategies to limit HIV-associated CNS disease. We have recently demonstrated that primary rhesus macaque astrocyte cultures are susceptible to infection in vitro by SIV/17E-Fr, a reproducibly neurovirulent molecular clone (2, 28, 55) but not the nonneurovirulent, lymphocyte-tropic molecular clone SIVmac239, which provided an excellent opportunity to study the viral determinants for SIV replication in astrocytes (37).

An important characteristic of neurovirulent viruses is the ability to infect key CNS cell types, each of which likely provides a unique contribution to the development of encephalitis. For HIV, differential specificity of Env proteins for either CCR5 or CXCR4 is a pivotal determinant of viral tropism; viruses that utilize CCR5 are generally macrophage tropic, those that use CXCR4 are lymphocyte tropic, and those that utilize both CCR5 and CXCR4 are considered dual tropic (6, 8, 9). For SIV, CCR5 is the main coreceptor utilized by Env proteins, irrespective of viral tropism (30). However, coreceptor specificity is not the only determinant of viral tropism, and several alterations within the SU and transmembrane (TM) portions of the Env protein of HIV (21, 31) and SIV (1, 3, 19, 20, 24, 35, 37) that are characteristic of viruses with the ability to replicate in nontraditional cellular targets and cause tissue-specific disease have been described. Several SIV tissue isolates from the brain and lung have unique Env features, in particular a reduced dependence on CD4, allowing these viruses to infect cells that express relatively low surface levels of CD4 and thereby perpetuating an expanded host cell tropism (14, 34).

CD4 is expressed at relatively low levels in cells of the CNS (38); thus, viruses with reduced dependence on CD4 likely have a selective advantage for replication in the brain. Notably, SIV/17E-Fr, in addition to being reproducibly neurovirulent, infects cells via a CD4-independent mechanism in vitro (14, 28, 40, 47). Specific amino acid differences in the Env and Nef proteins of SIV/17E-Fr compared to those of SIVmac239 that were critical for neurovirulence and the development of encephalitis were identified (1, 28, 47). In addition to conferring macrophage tropism, the amino acid changes within the SIV/17E-Fr Env facilitate replication in primary brain-derived microvessel endothelial cells (19), which have low surface expression of CD4 (14). Further, we have demonstrated previously that SIV/17E-Fr replication in primary astrocyte cultures is dependent upon the presence of a full-length Nef protein (37). Thus, small changes within two key viral genes, env and nef, allow a virus to expand its host cell range and infect cells in the brain.

In this study, we examined the role of specific amino acids in the SU and TM of Env in replication of SIV in primary rhesus macaque astrocytes. We report for the first time that in addition to the requirement of Nef for optimal replication in astrocytes, the presence of specific amino acids within the Env protein facilitates optimal replication. The SIV strain exhibiting optimal replication in astrocytes expressed a macrophage-tropic SU protein and a glycine (Gly) residue at amino acid 751 in the cytoplasmic tail of TM, in addition to a truncated cytoplasmic tail domain (CTD) of 50 amino acids. These data indicated that key amino acids within the cytoplasmic domain of TM enable SIV to replicate in primary astrocytes via a CD4-independent mechanism. The important role of CD4-independent entry of SIV into astrocytes was further supported by the selective replication of two distinct viral genotypes present within the SIV/DeltaB670 swarm previously shown to be CD4 independent. Together, these results strongly suggest that macrophage tropism is not sufficient for replication of SIV in primary astrocytes and that additional changes within the TM protein are necessary for the virus to enter and replicate in astrocytes via a CD4-independent mechanism.


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MATERIALS AND METHODS
 
Construction of SIV molecular clones. SIVmac239 open nef, SIV/17E-Fr, SIV/17E-Cl, SIV/3-11, SIV/3-7, and SIV/2-1 were constructed as previously described (19, 42). SIV/3-11RG was constructed by overlapping PCR with primers to introduce an A-to-G change at bp 8854, resulting in an Arg-to-Gly change at amino acid 751 in the TM protein (16). Mutation of bp 8854 to change the Arg residue to Gly confers no changes in the first open reading frame of tat, but does result in a conservative Lys-to-Arg substitution in the first open reading frame of rev. SIV/3-7 stop was constructed by site-directed mutagenesis by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) and primers 5'-AGCTCCTGGCCTTGACAGATAGAATATATTCTG and 5'-CAGAATATATTCTATCTGTCAAGGCCAGGAGCT with the template pLG 3-7 Nhe1-Blp1 fragment. After mutagenesis by PCR, the product was digested with NheI and BlpI (New England BioLabs, Beverly, Mass.) and ligated into full-length SIV/17E-Cl. The constructs were confirmed by DNA sequencing.

Virus stocks. Viral DNA was transfected into CEMX174 cells (a kind gift from James Hoxie), and virus was allowed to grow to high reverse transcriptase (RT) activity (greater than 90,000 cpm/ml) before being harvested. SIV/DeltaB670 was propagated in macaque primary blood lymphocytes and allowed to grow to high RT activity before being harvested. The virus-containing supernatants were filtered through a 0.45-µm-pore-size filter and pelleted through a 20% sucrose-TNE (40 mM Tris-HCl, 1 mM EDTA, and 150 mM NaCl) cushion for 2 h at 125,000 x g in a Sorvall Discovery 100SE ultracentrifuge. Virus stocks were resuspended in Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum, assayed for viral p27, and frozen at –80°C.

Culture and infection of primary macaque macrophages, PBL, and primary macaque astrocytes. Macaque primary blood-derived lymphocytes (PBL) were isolated from primary blood-derived mononuclear cells (PBMC) from an uninfected macaque via centrifugation through a Percoll gradient (Amersham Biosciences, Uppsala, Sweden). The cells were stimulated with phytohemagglutinin for 3 days and cultured at 2 x 106 cells/ml in RPMI supplemented with 10% fetal bovine serum, L-glutamine (2 mM), sodium pyruvate (2 mM), gentamicin (50 µg/ml), and recombinant human interleukin 2 (IL-2; 10 U/ml). Macrophages were obtained from macaque PBMC by culturing the cells for 5 days in macrophage differentiation medium (MDM; RPMI supplemented with 10% human AB-type serum, 100 U of recombinant human granulocyte-macrophage colony stimulating factor/ml, and 100 U of macrophage colony stimulating factor/ml [Genetics Institute, Cambridge, Mass.]).

For infection of PBL, 1.8 x 107 cells were pelleted, resuspended in 10% RPMI containing 300 ng of SIV p27 and IL-2, and allowed to incubate overnight at 37°C. The cells were washed three times with RPMI and resuspended in 10% RPMI supplemented with IL-2 at 2 x 106 cells/ml and plated in triplicate in six-well plates. Supernatants were collected every 2 days, and the medium was replaced with fresh medium. Supernatants were assayed for the presence of SIV p27 protein by using an SIV Core Antigen Assay kit (Coulter, Miami, Fla.).

Macrophages were infected with 50 ng of SIV p27 overnight at 37°C. Cells were washed six times with phosphate-buffered saline (Invitrogen) and cultured in MDM. The supernatants were collected every 2 days, and fresh 10% MDM was added. The supernatants were analyzed for the presence of virus as described above.

Primary rhesus macaque astrocytes (Cambrex, Walkersville, Md.) were cultured in Astrocyte Growth Medium (Cambrex) as previously described (37) at 37°C and 5% CO2. When astrocyte cultures reached 60 to 80% confluence, the medium was changed to Dulbecco's modified Eagle medium containing 10% fetal bovine serum, L-glutamine (2 mM), sodium pyruvate (2 mM), and gentamicin (50 µg/ml) 1 day prior to infection with SIV. Infections of primary macaque astrocytes were carried out in six-well plates coated with 0.01% poly-L-lysine. Cells were incubated with 50 ng of virus for 6 h and washed three times with Hanks buffered salt solution (HBSS) before fresh medium was added. The supernatants were collected every 5 days, and the cells were washed once with HBSS before fresh medium was again added. The supernatants were assayed for the presence of SIV p27 protein as described above.

For infection of the cell types described above, the virus input was normalized by p27 rather than the 50% tissue culture infective dose, because several of the clones analyzed in this study have changes within the TM protein and the TM has been shown to be important in regulating the fusion reaction. Because the 50% tissue culture infective dose measures the ability to induce fusion, we felt that normalizing input virus by p27 would more accurately reflect the virus's ability to replicate in a given cell type (23, 51).

Infections in the presence of recombinant soluble CD4. For experiments utilizing recombinant soluble CD4 (AIDS Reagent and Reference Program), virus stocks were incubated with 1 µg/ml at 37°C for 1 h prior to the infection of cells with 50 ng of SIV p27. Cells were washed three times with HBSS 6 h postinfection, and fresh medium with or without sCD4 was added. The supernatants were collected every 5 days and assayed for the presence of SIV p27 as described above.

Viral genotyping of SIV/DeltaB670. Primary macaque astrocytes were infected with 25,000 reverse transcriptase units of SIV/DeltaB670, and the cells were allowed to incubate until the virus reached high RT activity. Total cellular RNA was then isolated by using RNA-STAT (TelTest, Friendswood, Tex.). To determine the viral genotypes actively replicating within astrocyte cultures, total cellular RNA was subjected to two rounds of RT-PCR as previously described (2, 3) with the PCR primers 5'-AGGAATGCGACAATTCCCC-3' and 5'-CAGAGCGAAATGCAGTGATATTTATAC during the initial PCR, followed by amplification with 5'-CAGTCACAGAACAGGCAATAGA and 5'-CATCCCCTTGTGGAAAGTCC. The PCR product was cloned into the pSE-380 open vector with the SmaI (New England BioLabs) restriction enzyme and sequenced.


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RESULTS
 
Cellular tropism of SIV molecular clones. Previous studies from our laboratory identified a number of genetic determinants of neurovirulence. Recombinant SIV molecular clones that contained sequences derived from the lymphocyte-tropic, nonneurovirulent SIVmac239 and the macrophage-tropic, neurovirulent viral swarm SIV/17E-Br were generated (1, 47). Only one clone, SIV/17E-Fr, was found to be macrophage tropic in vitro and neurovirulent in vivo, and this clone contained amino acid changes within Env and Nef (from the parental SIVmac239) that were required for the neurovirulent phenotype (28) (see Fig. 2). This clone was also the only clone that replicated in macaque primary brain-derived microvessel endothelial cells (19). Another macrophage-tropic clone, SIV/17E-Cl, which contained the SU from SIV/17E-Br in the genetic background of SIVmac239, did not replicate in primary brain-derived endothelial cells and was not neurovirulent in vivo (19, 28) (see Fig. 2). We have previously demonstrated that SIVmac239 open nef does not replicate in astrocyte cultures, while SIV/17E-Fr replicates very efficiently (33).



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  		FIG. 2. Diagram of the genetic structure of a panel of SIV recombinant molecular clones used to examine the roles of specific TM sequences for replication in primary macaque lymphocytes, macrophages, and astrocytes. TM proteins diagrammed in black have an Arg residue at position 751, while those shown in gray have Gly at position 751. White bars indicate the presence of a stop codon in the TM CTD or in Nef (SIV/17E-Cl only). LTR, long terminal repeat.

To determine the requirements within the viral Env protein necessary for replication in primary macaque astrocytes, we analyzed the replication phenotype of four infectious molecular clones in primary astrocyte cultures and further compared their replication kinetics to those in primary lymphocytes and macrophages. While the growth properties of some of these clones in PBL and macrophages have been examined before (19), it was important to directly compare the in vitro cell tropism of the complete panel of SIV molecular clones in parallel cultures of PBL, macrophages, and astrocytes. Consistent with our previous findings, SIV/17E-Fr replicated efficiently in primary lymphocytes (Fig. 1A), primary macrophages (Fig. 1B), and primary astrocytes (Fig. 1C), while SIVmac239 established productive infection only in primary lymphocytes. Interestingly, the macrophage-tropic nonneurovirulent clone SIV/17E-Cl productively replicated in both lymphocytes (Fig. 1A) and macrophages (Fig. 1B) but did not establish productive infection in primary astrocyte cultures (Fig. 1C). As SIV/17E-Cl and SIV/17E-Fr differ only in their TM and Nef sequences, we analyzed SIV/2-1, which contains the lymphocyte-tropic env (SU only) and nef genes from SIVmac239 and the TM of SIV/17E-Fr. This clone established productive infection in primary lymphocytes but did not replicate in primary macrophages or astrocytes, indicating that the TM alone is also not sufficient to confer replication in astrocytes. Together, these data indicate that macrophage tropism is not sufficient for replication in astrocyte cultures and that specific amino acid sequences in the TM protein are required to establish productive infection in primary astrocyte cultures.



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  		FIG. 1. Replication kinetics of neurovirulent (SIV/17E-Fr) and nonneurovirulent (SIVmac239 and SIV/17E-Cl) SIV and SIV/2-1 (containing the TM of SIV/17E-Fr and the SU and Nef of SIVmac239) in primary rhesus macaque lymphocytes (A), macrophages (B), and astrocytes (C). Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.

As stated above, SIV/17E-Cl differs from SIV/17E-Fr in the TM and Nef proteins. Since we have previously demonstrated the importance of a full-length Nef protein for replication of SIV in primary astrocyte cultures (37), we focused on the TM in this study. There are only two sequence differences between the SIV/17E-Fr and SIV/17E-Cl TM proteins, these being an arginine (SIV/17E-Cl)-to-glycine (SIV/17E-Fr) change at amino acid 751 in the cytoplasmic tail domain (CTD) and the presence of a stop codon at amino acid 764 in the CTD of SIV/17E-Fr, which truncates the length of the CTD to 50 amino acids. SIV/17E-Cl expresses a full-length CTD of 166 amino acids. Interestingly, SIV replication in primary brain-derived endothelial cells mapped to the presence of glycine at position 751 in our previous studies (19). To differentiate the roles of these two changes in replication of SIV in astrocytes, we used a panel of infectious molecular clones containing different TM amino acid sequences (Fig. 2). The macrophage-tropic SIV/3-11 has the env gene of SIV/17E-Cl (full-length TM CTD with Arg at amino acid 751) with the open nef gene derived from SIVmac239 (19). The macrophage-tropic SIV/3-7 has the same env gene of SIV/3-11 but has the open nef gene derived from SIV/17E-Fr (19). SIV/3-11RG was constructed to test the role of the presence of glycine at amino acid 751 in the TM of SIV/17E-Fr in the context of a full-length cytoplasmic tail (Fig. 2). As SIV/17E-Fr also contains a truncated TM in addition to the R751G amino acid change, we constructed SIV/3-7stop, which contains the Arg residue at 751 in the context of a truncated TM cytoplasmic domain, to yield a cytoplasmic tail of 50 amino acids (Fig. 2). While truncation of the cytoplasmic tail domain found in SIV/17E-Fr was not an important determinant for macrophage tropism or replication in primary brain endothelial cells, truncated TM proteins are present in the brain during the course of SIV infection (20, 44; J. Clements, unpublished observations).

We compared the replication of the SIV molecular clones described above in PBL (Fig. 3A), primary rhesus macaque macrophages (Fig. 3B), and primary astrocytes (Fig. 3C) to distinguish the changes that confer lymphocyte and macrophage tropism from those that allow SIV to replicate in primary astrocytes. All of the molecular clones replicated efficiently in rhesus macaque PBL (Fig. 3A), indicating that all of the SIV Envs facilitated efficient entry and replication (Fig. 3A). Interestingly, while all molecular clones replicated productively in macrophages, SIV/3-11RG containing the Arg-to-Gly mutation in addition to the SU protein of SIV/17E-Fr replicated to higher titers in macrophages, indicating that the presence of Gly at position 751 in the TM protein may enhance SIV replication in macrophages (Fig. 3B). In addition, SIV/3-7stop, which contains a truncated TM protein, replicated efficiently in macrophages equal to the level of SIV/17E-Fr, indicating that a full-length cytoplasmic tail as well as the presence of Gly at position 751 in the cytoplasmic tail is preferred for optimal replication in macrophages.



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  		FIG. 3. Replication kinetics of SIV molecular clones containing mutations in TM in rhesus macaque primary lymphocytes (A), primary rhesus macaque macrophages (B), and primary rhesus macaque astrocytes (C). Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.

In contrast to replication in lymphocytes and macrophages, only a subset of the SIV molecular clones replicated efficiently in primary astrocytes (Fig. 3C). Importantly, molecular clones containing either the Gly residue at position 751 (SIV/3-11RG and SIV/17E-Fr) or the truncated cytoplasmic tail (SIV/3-7stop and SIV/17E-Fr) grew better in astrocytes than clones that expressed both full-length cytoplasmic tails and the original Arg residue found in SIVmac239 (SIV/3-11 and SIV/3-7) (Fig. 3C). In contrast to replication in macrophages, where Gly at position 751 optimized replication only in the context of an open TM protein, a Gly at 751 was preferred in astrocytes whether the TM was full-length or truncated. However, SIV/3-11RG and SIV/3-7stop did not replicate to the levels of SIV/17E-Fr, suggesting that although a Glycine residue at amino acid 751 and a truncated cytoplasmic tail can optimize replication of SIV in astrocytes, both mutations in the context of the same TM work best to facilitate optimal and efficient replication in primary macaque astrocytes. Collectively, these experiments clearly demonstrate a viral genetic basis for selective replication of SIV in astrocytes, as molecular clones that replicated efficiently in primary PBL or macrophages did not necessarily replicate efficiently in primary astrocyte cultures.

Role of CD4 independence in virus replication. The presence of specific sequences within the SIV cytoplasmic tail impact Env binding to cellular CD4 and chemokine coreceptors (16, 17, 27), notably by triggering conformational changes within the SU subunit that expose residues critical for coreceptor binding. Exposure of these critical residues enhances the binding between Env and the coreceptor and diminishes the affinity of Env for CD4 (25, 41). We considered the possibility that in the context of a macrophage-tropic SU protein, the Gly at position 751 or the truncated cytoplasmic tail enhances CD4-independent replication. Therefore, we examined the impact of treating virus with soluble CD4 (sCD4) to induce conformational changes in the Env protein prior to and throughout SIV infection of astrocytes. We have previously demonstrated that SIV utilizes CCR5 for entry by direct blocking of the chemokine receptor with RANTES that blocked replication in primary rhesus macaque astrocyte cultures. Astrocyte cultures express mRNA for CD4, but the cell surface expression is below the limit of detection of flow cytometry (37). Cultures of primary astrocytes were inoculated with SIV/17E-Fr, SIV/3-11, SIV/3-11RG, SIV/3-7, and SIV/3-7stop preincubated with 1 µg of recombinant human sCD4/ml. The addition of sCD4 greatly enhanced the infection of astrocytes by all of the molecular clones tested (compare Fig. 4A to Fig. 3C) but did not enhance the replication of viruses that contain Gly at position 751 in the context of a truncated CTD. Addition of sCD4 prior to and throughout infection allowed viruses that contain long cytoplasmic tails (SIV/3-11, SIV/3-11RG, and SIV/3-7) to replicate to higher levels than those expressing truncated CTD (SIV/17E-Fr and SIV/3-7stop), strikingly similar to the pattern of replication exhibited by these clones in macrophages (Fig. 3B). A comparison of the amount of virus replication in the presence of sCD4 at day 20 to the amount of virus produced without sCD4 (fold induction) demonstrated that clones expressing long cytoplasmic tails were greatly enhanced by sCD4, while those with the truncated CTD were only slightly enhanced, regardless of the presence of Arg or Gly at position 751 (Fig. 4B). These results suggest that the viral growth phenotypes demonstrated in astrocyte cultures without the presence of sCD4 (Fig. 3C) are likely the result of low-level or no expression of cell-surface CD4 on astrocytes.



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  		FIG. 4. Effect of soluble CD4 (sCD4) on replication of SIV infectious molecular clones in primary macaque cells. (A) SIV/17E-Fr, SIV/3-11, SIV/3-11RG, SIV/3-7, and SIV/3-7stop virus stocks were preincubated with 1 µg of sCD4/ml for 1 h at 37°C before 50 ng of virus was used to infect cells. The supernatants were collected every 5 days and assayed for viral p27 protein. (B) Induction of virus replication by sCD4 at day 20 postinoculation for the experiment shown in panel A. Fold induction is the log10 p27 value obtained with the sCD4/p27 value obtained without sCD4. (C) sCD4 facilitates replication of SIVmac239 in primary rhesus macaque astrocytes cultures. SIVmac239 was preincubated with sCD4, and the experiment was performed as described for panel A. (D) sCD4 does not enhance SIV/17E-Fr infection of primary rhesus macaque macrophages. Virus stocks were preincubated with 1 µg of sCD4/ml, and 50 ng of virus was used to infect primary macrophage cultures. The supernatants were collected every 2 days and analyzed for the presence of SIV p27 protein. For panels A, B, and C, data are from one of several independent experiments. Mock, mock-infected control.

Previous studies have demonstrated that the lymphocyte-tropic SIVmac239 is blocked at entry into macaque macrophages because of little to no surface expression of CD4 and that overexpression of CD4 in macaque macrophages allows SIVmac239 to establish productive infection (4, 33, 34). To analyze whether SIVmac239 is blocked at entry into astrocytes because of low levels of CD4 expression on the cell surface, we preincubated SIVmac239 virus stocks with sCD4 and infected macaque astrocyte cultures. In these experiments, SIVmac239 open nef was used to avoid the impact of the nef requirement for optimal replication in astrocytes. SIVmac239 open nef preincubated with sCD4 was able to establish productive replication in astrocyte cultures (Fig. 4C), although only to a fraction of the macrophage-tropic clones analyzed (compare Fig. 4A and C). This finding indicates that SIVmac239 is blocked at entry into primary astrocyte cultures because of little to no surface expression of CD4. The fact that SIVmac239 open nef was able to achieve only a low level of replication (compare Fig. 4A and C) indicates that there is likely a threshold amount of CD4 (either soluble or cellular surface bound) that is necessary to facilitate replication of SIVmac239 to levels of other virus strains in primary astrocytes.

Enhancement or blocking of SIV entry by sCD4 is dependent on the level of expression of CD4 on the target cell surface (46). To analyze whether sCD4 enhancement of replication is characteristic of SIV replication in other cell types, we also utilized sCD4-treated or untreated SIV/17E-Fr in viral growth assays in primary rhesus macrophages. SIV/17E-Fr infection was not enhanced and seemed to be slightly impaired during the early course of infection by sCD4 (Fig. 4D), a finding similar to those previously demonstrated for SIVmac316 infection of primary macaque macrophages (46). This finding is consistent with the notion that astrocytes express lower levels of CD4 than macrophages and again suggests that SIV strains with reduced dependence upon CD4 have a replicative advantage for replication in cells that express extremely low levels of CD4 (12).

Susceptibility of astrocyte cultures to other CD4-independent SIVs. SIV/DeltaB670 is a viral swarm consisting of more than 20 different genotypes exhibiting high sequence diversity from the SIVmac239-derived SIV/17E-Fr (2, 36, 52). When propagated in PBL, the relative frequencies of detectable SIV/DeltaB670 genotypes are 23% for Cl-12, 21% for Cl-2, and 13% for Cl-3, with eight other genotypes represented at less than 9% (2). When propagated in macrophages, the relative frequencies of detectable genotypes within SIV/DeltaB670 are 70% for Cl-12, 20% for Cl-2, and 10% for Cl-3 (2). Two of the genotypes within the SIV/DeltaB670 swarm, Cl-3 and Cl-12, express Env proteins that utilize CCR5 in a CD4-independent manner (15). Interestingly, Cl-3 and Cl-12 also replicate in the brains of infected macaques during acute infection (T. Babas, unpublished observations) in an accelerated, consistent SIV-macaque model (55). To determine whether SIV/DeltaB670 replicates in these primary CNS cells, astrocyte cultures were inoculated with SIV/DeltaB670 propagated in PBL, which replicated very efficiently (Fig. 5). To identify the genotypes of the SIV/DeltaB670 swarm that replicated in astrocytes, total cellular RNA was isolated from the infected astrocyte cultures, RT-PCR was performed, the V1 regions of the env genes were cloned, and DNA sequencing was performed as previously described (2, 3). Only Cl-3 and Cl-12, the previously described macrophage-tropic, CD4-independent genotypes, replicated in infected astrocyte cultures (sequencing data not shown). Because Cl-2 is represented at a higher frequency in the swarm inoculum than Cl-3 (21% versus 13% when grown in PBL), the observation that Cl-12 and Cl-3 but not Cl-2 are detectable in astrocytes cannot be accounted for by the relative frequencies in the inoculum. Further, since Cl-12, Cl-2, and Cl-3 replicate in primary PBL cultures and macrophage cultures, the ability of Cl-12 and Cl-3 to replicate in astrocytes strongly suggests the selection of these genotypes for replication in astrocytes rather than more efficient replication in general. Thus, these data are consistent with the hypothesis that macrophage tropism and CD4 independence are important determinants of replication in astrocytes.



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  		FIG. 5. Replication kinetics of SIV/DeltaB670 in primary astrocytes. Cells were incubated with SIV/DeltaB670, washed three times, and incubated at 37°C. Supernatants were collected every 5 days and assayed for the presence of SIV p27 protein. Data are from one of several independent experiments yielding similar results. Mock, mock-infected control.


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DISCUSSION
 
We have previously reported that full-length Nef is required for optimal replication in astrocytes (37). The studies presented here have expanded our analysis of the genetic requirements for SIV replication in primary rhesus macaque astrocytes. Our data demonstrate that macrophage tropism and specific amino acids within the TM protein that confer CD4 independence are critical determinants of SIV replication in astrocytes. Further, these experiments clarify the importance of amino acid differences between the nonneurovirulent SIVmac239 and the neurovirulent SIV/17E-Fr in Env and Nef that are characteristic of the neurovirulent phenotype (1, 28, 47). Specifically, these results strongly suggest that key residues within the TM portion of Env are required for CD4-independent Env protein function that in turn is necessary for the efficient infection of CNS cells, such as astrocytes, that express extremely low levels of CD4 (38).

Treatment of virus with sCD4 prior to infection of astrocytes greatly enhanced the replicative capacity of the molecular clones containing full-length cytoplasmic tails of TM, while clones expressing truncated TM proteins were only slightly enhanced by sCD4. This finding suggests that Envs with truncated TM proteins may exist in a partially triggered state, with key coreceptor binding sites already exposed to facilitate CD4-independent binding of CCR5. In contrast, it is likely that molecular clones containing full-length TM CTD do not have these key residues exposed within the coreceptor binding site; thus, sCD4 greatly enhanced their ability to replicate in astrocyte cultures. Sequence analyses of SIV present in multinucleated giant cells in the brains of SIV-infected macaques have revealed that truncated TM proteins are also found in vivo (20, 44; Clements, unpublished). Combined with the data presented here, these observations suggest that viruses expressing truncated TM proteins have an increased capacity to replicate in primary CNS cells and are thus likely important for neuropathogenesis.

The presence of Arg or Gly at position 751 did not impact the replication of viruses in the presence of sCD4, but clearly impacted replication in astrocytes without sCD4. Additionally, the presence of Gly at position 751 was preferred in macrophages in the context of a long TM. We compared the TM sequence of SIV/17E-Fr, Cl-3, and Cl-12 to look for similarities (data not shown), and while Cl-3 and Cl-12 have full-length TM cytoplasmic tails in addition to other sequence changes, both clones have a Gly residue at position 751, as does SIV/17E-Fr. Amino acid 751 does not lie within any discernible protein binding sequences or motifs, but sequence analysis illustrated that position 751 does lie within a region of the CTD that is particularly rich in glycine residues. SIV/17E-Fr and Cl-3 have Gly residues at 7 of 11 positions surrounding amino acid 751, indicating that this part of the CTD may be unstructured and flexible (26, 49). This increased flexibility likely translates into conformational changes within SU that facilitate the exposure of key coreceptor binding sites, independent of the truncation of the TM. Thus, the presence of Gly instead of Arg at position 751 may induce conformational changes within the SU that expose key coreceptor binding sites necessary for CD4-independent entry and replication, similar to those achieved with a truncated CTD. These results are in keeping with previous findings that mutations within TM impact CD4 independence (16, 17, 27).

We have previously demonstrated that astrocytes are nonproductively infected (express only Nef protein) during acute infection in our accelerated, consistent model of HIV-associated CNS disease (33). The macrophage-tropic, CD4-independent viruses Cl-3 and Cl-12 from the SIV/DeltaB670 swarm are the only viruses that replicate in the brain during acute infection prior to the development of CNS lesions, and thus these viruses are neuroinvasive (Babas, unpublished). In addition, these are the only SIV genotypes from the SIV/DeltaB670 swarm that replicated efficiently in astrocyte cultures in vitro, suggesting that Cl-3 and Cl-12 are likely the viruses found in astrocytes during acute infection. Because of the critical role of astrocytes in maintaining CNS homeostasis and inflammation, acutely infected astrocytes almost certainly play important roles in the pathogenesis of SIV-induced encephalitis. Early infection of astrocytes may contribute to the development of the innate immune response in the CNS that controls acute SIV replication (5, 10). Further, since astrocytes are the main producers of MCP-1 in the CNS and since increased MCP-1 levels in CSF precede and predict the development of moderate to severe CNS disease, infected astrocytes may also contribute to the infiltration and activation of macrophages associated with SIV encephalitis (11, 13, 18, 53).


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ACKNOWLEDGMENTS
 
We thank Brandon Bullock and Jesse DeWitt for their invaluable technical assistance and other members of the Retrovirus Laboratory for useful discussions of the data presented herein.

This work was supported by NIH grants NS47984, MH70306, NS35751, and NS38008 to J.E.C. and MH61189 to M.C.Z.


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FOOTNOTES
 
* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 733 N. Broadway, 819 BRB, Baltimore, MD 21205. Phone: (410) 955-9770. Fax: (410) 955-9823. E-mail: jclement{at}jhmi.edu. Back


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Journal of Virology, April 2005, p. 4944-4951, Vol. 79, No. 8
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