Vaccine protection by a triple deletion mutant of simian immunodeficiency virus

Twelve rhesus monkeys were vaccinated with SIVmac316 delta nef (lacking nef sequences), and 12 were vaccinated with SIVmac239 delta3 (lacking nef, vpr, and upstream sequences in U3). SIVmac316 and SIVmac239 differ by only eight amino acids in the envelope; these changes render SIVmac316 highly competent for replication in macrophages. Seventeen of the animals developed persistent infections with the vaccine viruses. Seven of the 24 vaccinated animals, however, developed infections that were apparently transient in nature. Six of these seven yielded virus from peripheral blood when tested at weeks 2 and/or 3, three of the seven had transient antibody responses, but none of the seven had persisting antibody responses. The 24 monkeys were challenged in groups of four with 10 rhesus monkey infectious doses of wild-type, pathogenic SIVmac251 at weeks 8, 20, and 79 following receipt of vaccine. None of the seven with apparently transient infections with vaccine virus were protected upon subsequent challenge. Analysis of cell-associated viral loads, CD4+ cell counts, and viral gene sequences present in peripheral blood in the remainder of the monkeys following challenge allowed a number of conclusions. (i) There was a trend toward increased protection with length of time of vaccination. (ii) Solid vaccine protection was achieved by 79 weeks with the highly attenuated SIV239 delta3. (iii) Solid long-term protection was achieved in at least two animals in the absence of complete sterilizing immunity. (iv) Genetic backbone appeared to influence protective capacity; animals vaccinated with SIV239 delta3 were better protected than animals receiving SIV316 delta nef. This better protection correlated with increased levels of the replicating vaccine strain. (v) The titer of virus-neutralizing activity in serum on the day of challenge correlated with protection when measured against a primary stock of SIVmac251 but not when measured against a laboratory-passaged stock. The level of binding antibodies to whole virus by enzyme-linked immunosorbent assay also correlated with protection.     

Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2

To study the effect of interleukin-2 (IL-2) on simian immunodeficiency virus (SIV) replication, pathogenesis, and immunogenicity, we replaced the nef gene of SIVmac239 by the IL-2 coding region. The virus, designated SIV-IL2, stably expressed high levels of IL-2 in cell culture. In comparison to SIVmac239, SIV-IL2 replicated more efficiently in peripheral blood mononuclear cells in the absence of exogenously added IL-2. To determine whether this growth advantage would be of relevance in vivo, four juvenile rhesus monkeys were infected with SIV-IL2 and four monkeys were infected with a nef deletion mutant of SIV (SIVdeltaNU). After a peak in the cell-associated viral load 2 weeks postinfection, the viruses could barely be isolated 3 to 7 months postinfection. Mean capsid antigen levels were higher in the SIV-IL2 group than in the nef deletion group 2 weeks postinfection. Viruses reisolated from the SIV-IL2-infected animals expressed high levels of IL-2 during the acute phase of infection. Deletions in the IL-2 coding region of SIV-IL2 were observed in two of the SIV-IL2-infected macaques 3 months postinfection. Urinary neopterin levels, a marker for unspecific immune stimulation, were higher in the SIV-IL2-infected macaques than in SIVdeltaNU-infected animals during the acute phase of infection. The SIV-specific T-cell-proliferative response and antibody titers were similar in both groups. Cytotoxic T cells directed against viral antigens were detected in all SIV-IL2-infected macaques and in two of the SIVdeltaNU-infected animals. Expression of IL-2 did not seem to alter the attenuated phenotype of nef deletion mutants fundamentally, although there might have been a slight increase in virus replication and immune stimulation during the acute phase of infection. Deletion of the viral IL-2 gene 3 months postinfection could be a consequence of a selective disadvantage due to local coexpression of viral antigen and IL-2 in the presence of an antiviral immune response.     

Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.     

Induction of MHC-IIDR expression on circulating CD8+ lymphocytes in macaques infected with SIVmac239 nef-open but not with its nef-deletion mutant

We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus strain SIVmac239 nef-open (239) or its nonpathogenic nef-deletion mutant (delta nef). In the longitudinal study, we found for the first time the induction of DR expression on CD8+ lymphocytes in 239-infected macaques. The induction of DR was in parallel with an increasing viral load and a decreasing CD4+ lymphocyte level. In the macaques with the high viral load and low CD4 level, a considerable proportion of the DR+CD8+ subpopulation was CD69+, indicating an activated state. On the other hand, no significant increase in the DR+CD8+ subpopulation level was observed in delta nef-infected macaques. These data indicate that the evaluation of activation markers such as DR and/or CD69 on circulating CD8+ cells may be valuable as a surrogate marker in the SIV-macaque model.     

Function of human immunodeficiency virus type 1 nef gene product in virus replication

We comparatively analyzed the replication kinetics of wild-type (wt) and nef mutant human immunodeficiency virus type 1 (HIV-1) in several CD4-positive cell lines, in order to clarify the molecular function of Nef protein. The delayed growth of nef mutant virus was observed at the initial stage of replication in all cell lines examined. This phenomenon was greatly amplified in the absence of vpu gene. In order to determine the infection stage in viral replication cycle which is specifically affected on virus replication rate in the presence of the Nef protein, we first examined the difference between wt and nef mutant viruses in the virus production rate from transfected cells, and found that the both viruses were produced with equal efficiency. This result showed that Nef protein could be dispensable for virion production. Therefore, early infection stages were focused by single-round infection assay, and the nef mutant virus was found to be much less infectious than wt virus. This indicated that the effect of Nef protein was exhibited in the early phase of a virus replication cycle, during viral adsorption to integration. By entry assay using wt and nef mutant virions, it was revealed that the Nef protein was required for efficient viral entry. These data suggest that the Nef protein might play a role in efficient incorporation of the Env protein into the virions, leading to enhanced viral infectivity.     

Direct demonstration of retroviral recombination in a rhesus monkey

Recombination may be an important mechanism for increasing variation in retroviral populations. Retroviral recombination has been demonstrated in tissue culture systems by artificially creating doubly infected cells. Evidence for retroviral recombination in vivo is indirect and is based principally on the identification of apparently mosaic human immunodeficiency virus type 1 genomes from phylogenetic analyses of viral sequences. We infected a rhesus monkey with two different molecularly cloned strains of simian immunodeficiency virus. One strain of virus had a deletion in vpx and vpr, and the other strain had a deletion in nef. Each strain on its own induced low virus loads and was nonpathogenic in rhesus monkeys. When injected simultaneously into separate legs of the same monkey, persistent high virus loads and declines in CD4+ lymphocyte concentrations were observed. Analysis of proviral DNA isolated directly from peripheral blood mononuclear cells showed that full-length, nondeleted SIVmac239 predominated by 2 weeks after infection. These results provide direct experimental evidence for genetic recombination between two different retroviral strains in an infected host. The results illustrate the ease and rapidity with which recombination can occur in an infected animal and the selection that can occur for variants generated by genetic recombination.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.     

SIV-associated nephropathy in rhesus macaques infected with lymphocyte-tropic SIVmac239

We examined the renal pathology and viral genetic changes following inoculation of six rhesus macaques with lymphocyte-tropic SIVmac239. Portions of the renal cortex were sieved into glomerular and tubulointerstitial (TI) fractions and examined for SIVmac sequences by PCR and for p27 core antigen. SIVmac sequences were detected in renal tissue from five of six macaques (three of five glomerular and five of five TI fractions were positive for SIV by PCR). Glomerulosclerosis (segmental and global) was evident in two macaques that were positive for env sequences in the glomerular fractions. Diffuse mesangial hyperplasia and matrix expansion were present in all three animals with glomerular SIV, as was an increase in glomerular collagen I and collagen IV. Tubulointerstitial inflammation was evident in all virus-inoculated macaques. The TI infiltration of CD68+ cells was most pronounced in the animals with SIVmac present in the glomerulus. All SIVmac-infected macaques exhibited increased glomerular deposition of IgM and to a lesser extent IgG, but no C3 or IgA was evident. Sequence analyses of the viral env gene (gp120) isolated from the glomerular and TI fractions of a macaque that developed glomerulopathy revealed the presence of specific viral variants in glomerular and TI fractions. In addition, chimeric viruses constructed with glomerular but not tubulointerstitial gp120 sequences were converted to a macrophage-tropic phenotype. These results indicate that infection by lymphocyte-tropic SIVmac239 is primarily associated with immunoglobulin deposition in the glomerulus and suggests that when glomerulosclerosis develops there is selection of viral variants that are macrophage tropic in nature.     

Temporal analyses of virus replication, immune responses, and efficacy in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Deltanef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Deltanef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10(5) to 10(7) RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Deltanef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Deltanef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Uberla, J. Virol. 71:2225-2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVDeltaNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVDeltaNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.     

Deletion of the nef gene abrogates the ability of SIV smmPBj to induce acutely lethal disease in pigtail macaques

Simian immunodeficiency virus (SIV) infection in macaque species is typically associated with the development of a progressive immunodeficiency disease, similar to human AIDS, resulting in death of animals in months to years after infection. In contrast, a variant virus, termed SIVsmmPBj, induces an acute disease in macaques, resulting in death in 5 to 14 days after infection. Previously, we have shown that several viral determinants contribute to the pathogenesis of this disease. The present study was undertaken to evaluate the role of Nef in the pathogenesis of SIVsmmPBj-induced acute disease. A molecular clone of SIVsmmPBj was generated that contains a deletion in the nef coding region (PBj6.6 delta nef). Virus derived from this molecular clone was tested with the parental virus, PBj6.6, in replication studies in pigtail macaque and rhesus macaque peripheral blood mononuclear cells (PBMCs). In general, PBj6.6 delta nef displayed markedly reduced replication abilities when compared with PBj6.6; the only exception being in stimulated pigtail macaque PBMCs, where replication kinetics were nearly identical. In addition, PBj6.6 delta nef was unable to induce the proliferation of peripheral blood mononuclear cells (PBMCs) in vitro, a unique characteristic of acutely pathogenic SIVsmmPBj. Inoculation of this virus into pigtail macaques resulted in infection, but did not result in any detectable acute disease. These studies suggest that Nef is an important viral determinant in the pathogenesis of SIVsmmPBj-induced disease, and further suggest that Nef plays a significant role in viral replication in vivo.     

Altered plaque formation by recombinant vaccinia virus expressing simian immunodeficiency virus Nef

The nef gene of primate lentiviruses encodes a myristoylated protein that is important for pathogenicity and the maintenance of high virus loads. A deletion in nef leads to a significant reduction of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. At the cellular and biochemical levels, Nef has been shown to down-regulate CD4 and major histocompatibility complex class I molecules and to interact with cellular protein kinases. The importance of these activities for Nef function remains uncertain. We have prepared vaccinia virus recombinants expressing different alleles of SIV nef. When grown on TK- 143 cells, recombinants constructed with the nef allele from SIVmac1A11 produced typical plaques while recombinants expressing the nef allele from SIVmac239-R1 gave rise to plaques with altered morphology. By using chimeric Nef proteins and site-directed mutagenesis, the amino acid responsible for altered plaque formation was mapped to a leucine at residue 211. In vitro phosphorylation of immunoprecipitates prepared from cells infected with the vaccinia virus recombinants resulted in labeled proteins of 62 and 90 kDa. The recombinants differed in the ability to stimulate phosphorylation, and the leucine at residue 211 was again found to be the determining amino acid. These results might help elucidate the role of nef in the pathogenesis of SIV.     

Diverse host responses and outcomes following simian immunodeficiency virus SIVmac239 infection in sooty mangabeys and rhesus macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10(5) to 10(7) RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8(+) T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4(+) T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4(+) T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.     

The isolation and characteristics of the green monkey lentivirus

We characterized the simian immunodeficiency virus isolated from Cercopithecus aethiops (subspecies C. a. pygerythrus) originating from Kenya. SIV was isolated and continuously produced with the MOLT4 clone 8 cell line and was designated as SIV-SU1. SIV-SU1 isolate replicated with high efficiency in MOLT4 clone 8, MT-2 with moderate efficiency in CEM x 174 and with poor efficiency in HUT-78, U937, C8166. The infection of MT-2, C8166 and HUT-78 resulted in extensive cell killing. Western blotting of purified preparations of SIV-SU1 revealed viral proteins of 130, 68, 55, 41, 24, 17 kDa. Cross-reactivity of SIV-SU1 proteins with HIV-1, HIV-2, SIVmac, SIVsm, SIVmnd was studied by radioimmunoprecipitation assay. The most extensive cross-reactivity was observed with SIVmac. Total cellular DNA from chronically infected cells was hybridized to SIVagm266 DNA probes. Detection of cross-hybridizing DNA sequences required very low stringency, and the restriction endonuclease fragmentation pattern of SIV-SU1 differed from other SIVs.     

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.