Regulation of SIV mac 239 basal long terminal repeat activity and viral replication in macrophages: functional roles of two CCAAT/enhancer-binding protein beta sites in activation and interferon beta-mediated suppression.

CCAAT/enhancer-binding protein (C/EBP) beta and C/EBP sites in the HIV-1 long terminal repeat (LTR) are crucial for HIV-1 replication in monocyte/macrophages and for the ability of interferon beta (IFN beta) to inhibit ongoing active HIV replication in these cells. This IFN beta-mediated down-regulation involves induction of the truncated, dominant-negative isoform of C/EBP beta referred to as liver-enriched transcriptional inhibitory protein (LIP). Although binding of the C/EBP beta isoform to C/EBP sites in the simian immunodeficiency virus (SIV) LTR has previously been examined, the importance of these sites in core promoter-mediated transcription, virus replication, IFN beta-mediated regulation, and the relative binding of the two isoforms (C/EBP beta and LIP) has not been investigated. Here, we specifically examine two C/EBP sites, JC1 (-100 bp) and DS1 (+134 bp), located within the minimal region of the SIV LTR, required for core promoter-mediated transcription and virus replication in macrophages. Our studies revealed that the JC1 but not DS1 C/EBP site is important for basal level transcription, whereas the DS1 C/EBP site is imperative for productive virus replication in primary macrophages. In contrast, either JC1 or DS1 C/EBP site is sufficient to mediate IFN beta-induced down-regulation of SIV LTR activity and virus replication in these cells. We also characterized the differential binding properties of C/EBP beta and LIP to the JC1 and DS1 sites. In conjunction with previous studies from our laboratory, we demonstrate the importance of these sites in virus gene expression, and we propose a model for their role in establishing latency and persistence in macrophages in the brain.

Minocycline attenuates HIV infection and reactivation by suppressing cellular activation in human CD4+ T cells.

Treatment of human immunodeficiency virus (HIV) infection with highly active antiretroviral therapy (HAART) is effective but can be associated with toxic effects and is expensive. Other options may be useful for long-term therapy. The immunomodulatory antibiotic minocycline could be an effective, low-cost adjunctive treatment to HAART. Minocycline mediated a dose-dependent decrease in single-cycle CXCR4-tropic HIV infection and decreased viral RNA after infection of CD4+ T cells with HIV NL4-3. Reactivation from latency was also decreased in a primary CD4+ T cell-derived model and in resting CD4+ T cells from HIV-infected patients. Minocycline treatment resulted in significant changes in activation marker expression and inhibited proliferation and cytokine secretion of CD4+ T cells in response to activation. This study demonstrates that minocycline reduces HIV replication and reactivation and decreases CD4+ T cell activation. The anti-HIV effects of minocycline are mediated by altering the cellular environment rather than directly targeting virus, placing minocycline in the class of anticellular anti-HIV drugs.

Mechanisms of minocycline-induced suppression of simian immunodeficiency virus encephalitis: inhibition of apoptosis signal-regulating kinase 1.

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) can lead to cognitive dysfunction, even in individuals treated with highly active antiretroviral therapy. Using an established simian immunodeficiency virus (SIV)/macaque model of HIV CNS disease, we previously reported that infection shifts the balance of activation of mitogen-activated protein kinase (MAPK) signaling pathways in the brain, resulting in increased activation of the neurodegenerative MAPKs p38 and JNK. Minocycline treatment of SIV-infected macaques reduced the incidence and severity of SIV encephalitis in this model, and suppressed the activation of p38 in the brain. The purpose of this study was to further examine the effects of minocycline on neurodegenerative MAPK signaling. We first demonstrated that minocycline also decreases JNK activation in the brain and levels of the inflammatory mediator nitric oxide (NO). We next used NO to activate these MAPK pathways in vitro, and demonstrated that minocycline suppresses p38 and c-Jun N-terminal kinase (JNK) activation by reducing intracellular levels, and hence, activation of apoptosis signal-regulating kinase 1 (ASK1), a MAPK kinase capable of selectively activating both pathways. We then demonstrated that ASK1 activation in the brain during SIV infection is suppressed by minocycline. By suppressing p38 and JNK activation pathways, which are important for the production of and responses to inflammatory mediators, minocycline may interrupt the vicious cycle of inflammation that both results from, and promotes, virus replication in SIV and HIV CNS disease.

From mice to macaques--animal models of HIV nervous system disease.

Lentiviral diseases of animals have been recognized for over a century, long before HIV was recognized as the cause of AIDS. All lentiviruses cause neurological disease and productive virus replication in the CNS occurs exclusively in cells of macrophage lineage. The ability to molecularly engineer the inoculum virus, to sample the brain at many different time points from acute through terminal infection and to correlate in vivo with in vitro findings are significant advantages of animal models of HIV CNS disease. The lentiviruses can be divided into two pathogenetic groups--those that cause immunosuppression, including the lentiviruses of humans (HIV), non-human primates (SIV), cats (FIV), and cattle (BIV), and those that cause immunoproliferation, including the lentiviruses of horses (EIAV), sheep (OvLV) and goats (CAEV). Despite extensive study, no rodent lentivirus has been identified, prompting development of alternate strategies to study lentiviral pathogenesis using rodents. The immunosuppressive lentiviruses most closely recapitulate the disease manifestations of HIV infection, and both SIV and FIV have contributed significantly to our understanding of how HIV causes both central and peripheral nervous system disease.

The SIVmac239 Pr55Gag isoform, SIV p43, suppresses proteolytic cleavage of Pr55Gag.

In human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) the gag gene encodes the precursor polyprotein Pr55Gag, which is cleaved by the viral protease to produce the major structural proteins. Recently, it has been shown that HIV and SIV gag RNAs contain internal ribosome entry sites (IRESs) that mediate translation of Pr55Gag [Pr57Gag in HIV type 2 (HIV-2)] isoforms. Previously, we demonstrated that SIVmac239 p43(-), a mutant that does not express the Pr55Gag isoform, SIV p43, replicates more efficiently than wild-type (WT) SIVmac239 in cell culture. In this study, we characterize SIVmac239 p43(-) virion production and demonstrate that, in the absence of SIV p43, cleavage of Pr55Gag is increased in budded virions, resulting in a higher percentage of mature particles. Additionally, intracellular cleavage of Pr55Gag is increased in SIVmac239 p43(-), suggesting that SIV p43 suppresses premature cleavage of Pr55Gag by the viral protease.

CUGBP1 is required for IFNbeta-mediated induction of dominant-negative CEBPbeta and suppression of SIV replication in macrophages.

Productive HIV replication in the CNS occurs very early after infection, yet HIV-associated cognitive disorders do not typically manifest until the development of AIDS, suggesting that mechanisms exist in the CNS to control HIV replication and associated virus-induced pathological changes during the acute and asymptomatic stages of disease. Using an established SIV/macaque model of HIV dementia, we recently demonstrated that the mechanisms regulating virus replication in the brain at these stages involve the production of IFNbeta, which induces the truncated, dominant-negative isoform of C/EBPbeta, also referred to as LIP (liver-enriched transcriptional inhibitory protein). Alternative translation of C/EBPbeta mRNA and increased production of LIP can be mediated by CUGBP1 (CUG-repeat RNA-binding protein 1). Because IFNbeta induces the inhibitory C/EBPbeta in macrophages, we considered the possibility that IFNbeta signaling regulates the activity of CUGBP1, resulting in increased expression of LIP and suppression of SIV replication. In this study, we report that IFNbeta induces LIP and suppresses active SIV replication in primary macrophages from rhesus macaques. Further, we demonstrate that IFNbeta induces the phosphorylation of CUGBP1 and the formation of CUGBP1-C/EBPbeta mRNA complexes in the human monocytic U937 cell line. Finally, we demonstrate that CUGBP1 is not only required for IFNbeta-mediated induction of LIP but also for IFNbeta-mediated suppression of SIV replication. These results suggest that CUGBP1 is a previously unrecognized downstream effector of IFNbeta signaling in primary macrophages that likely plays a pivotal role in innate immune responses that control acute HIV/SIV replication in the brain.

Sequence variation in the CC-chemokine ligand 2 promoter of pigtailed macaques is not associated with the incidence or severity of neuropathology in a simian immunodeficiency virus model of human immunodeficiency virus central nervous system disease.

Increased expression of CC-chemokine ligand 2 (CCL2) in the cerebrospinal fluid (CSF) and brain is consistently observed in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) central nervous system (CNS) disease. The molecular basis for the correlation between increased expression of CCL2 and HIV neuropathogenesis has been linked to a polymorphism at -2578 in the promoter of human CCL2, which was reported to influence the rate of progression to acquired immunodeficiency syndrome (AIDS) and the predisposition of HIV-infected individuals to develop HIV-associated dementia. However, because the rate of neurological deterioration essentially parallels the progression of immunosuppression, it is inherently difficult to uncouple the influence of this polymorphism on increased progression to AIDS from increased propensity to develop CNS complications. To further investigate the correlation between CCL2 and HIV/SIV CNS disease, the authors sequenced the CCL2 promoter of 29 pigtailed macaques examined in their accelerated and consistent SIV model in which all infected macaques develop AIDS but only 69% developed moderate/severe CNS lesions. Sequence analysis identified 39 sites of nucleotide variation in the pigtailed macaque CCL2 promoter/enhancer regions, with the resulting consensus sequence aligning with 94.7% homology to the human CCL2 promoter. After genetic analyses, no variation was found to correlate with the incidence or severity of CNS lesions or with levels of CCL2 in plasma or CSF. These findings suggest that the determinants of neuropathogenesis in this SIV model are distinct from variation in these regions of the CCL2 promoter.

Protein kinase CK2 phosphorylates the Nef protein from a neurovirulent simian immunodeficiency virus.

The Nef protein of Human Immunodeficiency Virus (HIV) and Simian Immunodeficiency Virus (SIV) is a pluripotent accessory protein that plays a critical role in disease progression. One analogous characteristic of Nef proteins from SIV and HIV is the ability to associate with cellular kinases. We have previously reported that the Nef protein from a macrophage-tropic neurovirulent SIV clone, SIV/17E-Fr, is associated with an unknown kinase activity that is distinct from the p21-associated kinase that interacts with SIVmac239 Nef. Using site-directed mutagenesis and kinase-specific inhibitors, we have identified this kinase as the ubiquitous serine/threonine kinase, protein kinase CK2.

Mechanism for the establishment of transcriptional HIV latency in the brain in a simian immunodeficiency virus-macaque model.

BACKGROUND: The brain is considered to be a reservoir of latent human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We examined the mechanism by which innate immune responses contribute to the establishment of this reservoir. METHODS: Gene-specific RNA and DNA were quantitated using real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using Western blot analysis. Binding to and regulation of the SIV long terminal repeat (LTR) was examined using electrophoretic mobility shift assay, luciferase reporter constructs, and chromatin immunoprecipitation assay. RESULTS: Interferon-beta (IFN-beta) and myxovirus A (MxA) mRNA are produced in the brain during acute SIV infection. IFN-beta both suppresses SIV LTR activity and induces expression of the dominant-negative isoform of CCAAT/enhancer-binding protein-beta (C/EBP-beta). C/EBP-beta and its dominant-negative isoform respectively enhance and suppress histone acetylation at the SIV LTR and are present at the SIV LTR in vivo. SIV DNA persists when viral RNA is undetectable in the brain, and activation of the LTR is suppressed at the level of histone acetylation. CONCLUSION: Innate immune responses to virus infection that suppress acute virus replication in the brain also facilitate transcriptional latency of SIV. These data provide the first mechanistic model of HIV latency in the brain.

An internal ribosome entry site promotes translation of a novel SIV Pr55(Gag) isoform.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

Longitudinal analysis of simian immunodeficiency virus (SIV) replication in the lungs: compartmentalized regulation of SIV.

BACKGROUND: Before the onset of AIDS, replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in the lungs is considered to be latent. When and how virus replication is controlled in the lungs is unclear. In the present study, we examine virus replication in the lungs and in cells recovered from bronchoalveolar lavage (BAL) samples in a comprehensive, longitudinal analysis of an SIV/macaque model. METHODS: Gene-specific RNA and DNA were quantitated by polymerase chain reaction (PCR) and by real-time reverse-transcription PCR (RT-PCR). Alveolar macrophages were isolated using Dynabeads CD14 (Invitrogen). Expression of CCAAT/enhancer-binding protein beta (C/EBP beta ) isoforms was examined by Western blot analysis. RESULTS: SIV replication occurred in the lungs during acute infection and correlated with plasma viral load. Innate immune responses involving interferon- beta and the dominant-negative isoform of C/EBP beta were induced at this time. SIV RNA expression was suppressed in the lungs during asymptomatic infection, when no correlation existed with plasma viral load until SIV RNA levels rebounded again during late-stage disease. Modulation of viral RNA levels in BAL cells reflected RNA levels in lung tissue throughout each phase of infection. CONCLUSION: Quantitation of SIV RNA in BAL cells provides a consistent surrogate assessment of virus replication in lung tissue. Innate immune responses contribute to compartmentalized suppression of acute SIV replication in the lungs.

Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus.

The lentiviral Gag polyprotein (Pr55(Gag)) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55(Gag) is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55(Gag) and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55(Gag), several CA-containing cleavage intermediates of Pr55(Gag), and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55(Gag) can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55(Gag) and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55(Gag) and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.

CD4-independent entry and replication of simian immunodeficiency virus in primary rhesus macaque astrocytes are regulated by the transmembrane protein.

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.

Conserved serines in simian immunodeficiency virus capsid are required for virus budding.

The simian immunodeficiency virus (SIV) capsid protein (CA), a constituent of the Pr55Gag polyprotein, is phosphorylated in virions but not in virus-producing cells (Rue, S.M., Roos, J.W., Tarwater, P.M., Clements, J.E., Barber, S.A., 2005. Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus. J. Virol. 79 (4), 2484-2492.). Using phosphoamino acid analysis of CA, we show that serine is the primary phosphate acceptor. A series of substitution mutants of serines in the CA domain of Pr55Gag were constructed in the infectious viral clone SIVmac239. These virus mutants were examined for defects in virus replication and virion infectivity, release, and morphology, as well as alterations in phosphorylation of CA-containing proteins. Although the virus mutants exhibited a number of replication defects, none of these defects could be directly attributed to aberrant CA phosphorylation. A novel defect was a block in early budding, which was common among several virus mutants with substitutions in the CA N terminus. Together, these results indicate that certain residues in the CA N terminus are crucial for early budding events.

Innate immune responses and control of acute simian immunodeficiency virus replication in the central nervous system.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) can invade the central nervous system (CNS) during acute infection but virus replication is apparently controlled because clinical and pathological manifestations of CNS disease in HIV/SIV-infected individuals usually present later in infection, coincident with immunosuppression and acquired immuno-deficiency syndrome (AIDS). Using an established SIV/macaque model of HIV dementia, the authors recently demonstrated that acute virus replication is down-regulated (to undetectable viral RNA levels) in the brain, but not the periphery, as early as 21 days post inoculation (p.i.). Viral DNA levels in the brain remain constant, suggesting that infected cells persist in the CNS and that replication is inhibited largely at a transcriptional level. In vitro, active replication of HIV in macrophages can be inhibited by treatment with interferon (IFN)beta via a mechanism involving induction of a dominant-negative form of the transcription factor C/EBP (CCAAT/enhancer-binding protein)beta. Because macrophages are the primary cell types infected with HIV/SIV in the CNS and HIV replication in macrophages requires C/EBP sites within the viral long terminal repeat (LTR), the authors considered the possibility that suppression of C/EBP-dependent transcription contributes to the mechanism by which acute HIV/SIV replication is inhibited in the CNS. Here, the authors report that IFNbeta can also inhibit ongoing SIV replication in macaque macrophages in vitro. Further, the authors demonstrate that IFNbeta levels in the brain increase between 7 and 21 days p.i. in parallel with increased expression of the dominant-negative isoform of C/EBPbeta. These results suggest that innate immune responses involving IFNbeta may contribute to the mechanism(s) controlling acute SIV replication in the CNS.

Dysregulation of mitogen-activated protein kinase signaling pathways in simian immunodeficiency virus encephalitis.

Central nervous system (CNS) disease is a frequent complication of human immunodeficiency virus (HIV)-1 infection. Identification of cellular mechanisms that control virus replication and that mediate development of HIV-associated neuropathology will provide novel strategies for therapeutic intervention. The milieu of the CNS during HIV infection is extraordinarily complex because of infiltration of inflammatory cells and production of chemokines, cytokines, and neurotoxic molecules. Cells in the CNS must integrate signaling pathways activated simultaneously by products of virus replication and infiltrating immune cells. In this study, we examined activation of mitogen-activated protein kinases (MAPKs) in the CNS of simian immunodeficiency virus-infected macaques during acute, asymptomatic, and terminal infection. We demonstrate that significantly increased (P < 0.02) activation of ERK MAPK, typically associated with anti-apoptotic and neuroprotective pathways, occurs predominantly in astrocytes and immediately precedes suppression of virus replication and macrophage activation that occur after acute infection. In contrast, significantly increased activation of proapoptotic, neurodegenerative MAPKs JNK (P = 0.03; predominantly in macrophages/microglia), and p38 (P = 0.03; predominantly in neurons and astrocytes) after acute infection correlates with subsequent resurgent virus replication and development of neurological lesions. This shift from classically neuroprotective to neurodegenerative MAPK pathways suggests that agents that inhibit activation of JNK/p38 may be protective against HIV-associated CNS disease.

Hydrogen bonding at a conserved threonine in lentivirus capsid is required for virus replication.

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55(Gag) polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)(CA), that is requisite for viral replication. Replacement of T(47)(CA) in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)(CA) forms several hydrogen bonds with a proximal aspartate, D(50)(CA). This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)(CA) and D(50)(CA) is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.

Visna virus-induced activation of MAPK is required for virus replication and correlates with virus-induced neuropathology.

It is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression of gag and env, but not rev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.

Expression of simian immunodeficiency virus (SIV) nef in astrocytes during acute and terminal infection and requirement of nef for optimal replication of neurovirulent SIV in vitro.

As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.