SIVmac239 Nef Research Articles

SIV/HIV nef Recombinant Virus (SHIVnef) Produces Simian AIDS in Rhesus Macaques

The simian immunodeficiency virus (SIV) nef gene is an important determinant of viral load and acquired immunodeficiency syndrome (AIDS) in macaques. A role(s) for the HIV-1 nef gene in infection and pathogenesis was investigated by constructing recombinant viruses in which the nef gene of the pathogenic molecular clone SIVmac239 nef was replaced with either HIV-1sf2nef or HIV-1sf33nef. These chimeras, designated SHIV-2nef and SHIV-33nef, expressed HIV-1 Nef protein and replicated efficiently in cultures of rhesus macaque lymphoid cells. In two SHIV-2nef-infected juvenile rhesus macaques and in one of two SHIV-33nef-infected juvenile macaques, virus loads remained at low levels in both peripheral blood and lymph nodes in acute and chronic phases of infection (for >83 weeks). In striking contrast, the second SHIV-33nef-infected macaque showed high virus loads during the chronic stage of infection (after 24 weeks). CD4+ T-cell numbers declined dramatically in this latter animal, which developed simian AIDS (SAIDS) at 47–53 weeks after inoculation; virus was recovered at necropsy at 53 weeks and designated SHIV-33Anef. Sequence analysis of the HIV-1sf33nef gene in SHIV-33Anef revealed four consistent amino acid changes acquired during passage in vivo. Interestingly, one of these consensus mutations generated a tyr-x-x-leu (Y-X-X-L) motif in the HIV-1sf33 Nef protein. This motif is characteristic of certain endocytic targeting sequences and also resembles a src-homology region-2 (SH-2) motif found in many cellular signaling proteins. Four additional macaques infected with SHIV-33Anef contained high virus loads, and three of these animals progressed to fatal SAIDS. Several of the consensus amino acid changes in Nef, including Y-X-X-L motif, were retained in these recipient animals exhibiting high virus load and disease. In summary, these findings indicate that the SHIV-33Anef chimera is pathogenic in rhesus macaques and that this approach, i.e., construction of chimeric viruses, will be important for analyzing the function(s) of HIV-1 nef genes in immunodeficiency in vivo, testing antiviral therapies aimed at inhibiting AIDS, and investigating adaptation of this HIV-1 accessory gene to the macaque host.

The Conserved Core of Human Immunodeficiency Virus Type 1 Nef Is Essential for Association with Lck and for Enhanced Viral Replication in T-Lymphocytes

The Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. We report the functional interactions of the Nef proteins with Src kinases. The Nef proteins from HIV-1 and SIV bind to Lck as well as Hck, Lyn, and Fyn. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. In addition, we show that SIV and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not phosphorylate HIV-1 Nef. These data suggest that the association of Nef and Lck is central to the enhanced viral replication of HIV-1 and SIV in T-lymphocytes.

Effects of SIVmac infection on peripheral blood CD4+CD8+ T lymphocytes in cynomolgus macaques.

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.

Selection of the R17Y Substitution in SIVmac239 Nef Coincided with a Dramatic Increase in Plasma Viremia and Rapid Progression to Death

Three rhesus macaques were infected with an SIVmac239 variant containing substitutions of 73/74PA→ED and 204D→R in Nef that disrupted the ability of Nef to downregulate CD4 surface expression. One of these animals, Mm8155, rapidly progressed to AIDS and died 21 weeks postinfection. During the final 5 weeks of infection, the levels of viral RNA and of p27 antigenemia were about 100-fold higher than usually observed in SIVmac239 infection. Postmortem examination revealed giant cell disease of the lymph nodes and the gastrointestinal tract, opportunistic infections, and a severe chronic enteritis. The majority of proviruses in spleen, kidney, and lymph nodes, and almost 100% of the viral RNA sequences, contained mutations of CGA→TAT in codon 17 ofnef,predicting a change of 17R→Y. The appearance of this substitution, which has recently been shown to confer the phenotype of the acutely pathogenic SIVpbj14, coincided with the dramatic increase in viral load and rapid progression to fatal disease. In comparison, reversions of 204R→D and changes of 72-74NED→DKD, which restored the ability of Nef to downregulate CD4, were already selected earlier in infection. Similarly to SIVpbj14, virus reisolated at late time points from Mm8155 replicated efficiently in unstimulated monkey lymphocytes. The Y17 substitution was not detected in 14 additional SIVmac239-infected macaques at the time of AIDS-related death or in the two slowly progressing animals initially infected with the same Nef variant. Although infection of macaques with SIV is commonly used as an animal model for HIV-1 infection in humans, this is only the second example for the emergence of an acutely lethal SIVmac Nef variant.

A Novel Kinase Activity Associated with Nef Derived from Neurovirulent Simian Immunodeficiency Virus

The Nef proteins of Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) have been shown to associate with several cellular kinases. Further, the ability of SIVmac239 Nef to associate with a p21-activated kinase (PAK)-related kinase has been correlated with pathogenic progression to AIDS in rhesus macaques. Because the ability of Nef to associate with the PAK-related kinase is viral isolate dependent, we reasoned that viral isolates derived from distinct physiological locations may encode Nef proteins that exhibit distinct kinase association profiles. In this study, we compared kinase activities associated with Nef proteins derived from the prototypic lymphocyte-tropic SIVmac239 and a macrophage-tropic, neurovirulent clone, SIV/17E-Fr. Our findings not only support previous studies that have documented the association of SIVmac239 Nef with a PAK-related kinase and a Nef-associated kinase complex (NAKC) but describe a novel serine kinase activity detectable only in conjunction with the Nef protein derived from the neurovirulent clone, SIV/17E-Fr. The latter Nef protein does not associate with PAK, and unlike PAK or NAKC, this novel kinase activity is enhanced in association with nonmyristoylated forms of Nef and can utilize both ATP and GTP as phosphodonors. We also show that at least one substrate for the kinase is Nef itself and demonstrate that the SIV/17E-Fr Nef protein is phosphorylated in SIV-infected cells. These results suggest that the ability to associate with cellular kinases in general may be a conserved feature of Nef, but particular kinase/Nef associations may evolve with changes in the host environment concomitant with viral spread.

Mice transgenic for simian immunodeficiency virus nef are immunologically compromised.

An intact nef gene is essential for rapid development of immunodeficiency in human immunodeficiency virus and simian immunodeficiency virus infections. To assess the role of nef in the immune response, mice transgenic for SIV nef were constructed and the humoral and cellular immune response to herpes simplex virus type-1 (HSV-1), measured. Mice transgenic for SIVmac239 nef exhibited a significantly increased mortality rate when challenged with HSV-1 and also showed unusual antibody kinetics in response to viral challenge. During a 32-week period following exposure to HSV, it was noted that IgG subclass titers continued to rise in the nef+ animals, while titers of nef- animals decreased. Additionally, following secondary challenge with HSV, nef- mice had a significantly greater rise in HSV-neutralizing antibody titers than nef+ mice. A decreased proliferative response to the T cell mitogen, PHA, was noted in the nef+ animals. These results suggest that the presence of nef+ is sufficient to induce immune dysfunction.

A genetic and viral load analysis of the simian immunodeficiency virus during the acute phase in macaques inoculated by the vaginal route.

A comparative genetic analysis of SIV-infected female macaques during the first 120 days postinfection was undertaken. The same dose of a macaque-passaged SIVmac239(nef open) was administered to three macaques intravenously (i.v.) and to three macaques intravaginally (i.VAG). Clinical outcomes observed ranged from rapid to nonprogression, while two of the i.v.-infected macaques developed an uncommon hindleg paresis. Analysis of viral load (bDNA assay) determined that both i.v.- and i.VAG-infected macaques had comparable high viral loads at the observed viral peak of 14 days postinfection. A study of viral quasispecies diversity by the heteroduplex mobility assay indicated that (1) the i.v.-infected macaques had a highly heterogeneous quasispecies population similar to the infecting viral stock; and (2) in two of three i.VAG-infected macaques multiple viral genotypes (minimum, three or four) were observed in blood and lymph tissues at early times postinfection, which indicated that limited numbers of viral variants crossed the vaginal mucosa and established infection. Therefore, the route of infection can clearly influence early viral selection and diversity. In addition, a third i.VAG-infected macaque, which was a rapid progressor, did not seroconvert and progressed to AIDS in 120 days. This macaque exhibited a high viral load and heterogeneous quasispecies. These data demonstrate differences in the quasispecies complexity associated with route of infection and rate of disease progression.

Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques.

The nef gene of simian immunodeficiency virus (SIV) is essential for high viral load and induction of AIDS in rhesus monkeys. A mutant form of the SIVmac239 Nef, which contains changes in a putative SH3-binding domain (amino acids 104 and 107 have been changed from PxxP to AxxA), does not associate with cellular serine/threonine kinases, but is fully active in CD4 downregulation and associates with the cellular tyrosine kinase Src. Infection of two rhesus macaques with SIVmac239 containing the mutant AxxA-Nef caused AIDS and rapid death in both animals. No reversions were observed in the majority of nef sequences analyzed from different time points during infection and from lymphatic tissues at the time of death. Our findings indicate that the putative SH3-ligand domain in SIVmac Nef and the association with cellular serine/threonine kinases are not important for efficient replication and pathogenicity of SIVmac in rhesus macaques.

Requirements for lymphocyte activation by unusual strains of simian immunodeficiency virus.

When residues 17 and 18 in nef of simian immunodeficiency virus strain SIVmac239 were changed from RQ to YE, the resultant virus was able to replicate in peripheral blood mononuclear cell cultures without prior lymphocyte activation and without the addition of exogenous interleukin-2, caused extensive lymphocyte activation in these cultures, and produced an acute disease in rhesus and pigtail macaques (Z. Du, S. M. Lang, V. G. Sasseville, A. A. Lackner, P. 0. Ilyinskii, M. D. Daniel, J. U. Jung, and R. C. Desrosiers, Cell 82:665-674, 1995). These properties are similar to those of the acutely lethal pathogen SIVpbj14 but dissimilar to those of the parental SIVmac239. We show here that the single change of R to Y at position 17 in nef of SIVmac239 is sufficient to confer the full, unusual phenotype. Conversely, the lymphocyte-activating properties of SIVpbj14 were lost by the single change of Y to R at position 17 of nef. The change of R17F or Q18E in SIVmac239 nef did not confer the unusual in vitro properties. Since SIVpbj14 has a duplication of the NF-kappaB binding sequence in the transcriptional control region, we also constructed and tested strains of SIVmac239/Rl7Y with zero, one, and two NF-kappaB binding elements. We found no difference in the properties of SIVmac239/R17Y, either in cell culture or in vivo, whether zero, one, or two NF-kappaB binding sites were present. Thus, tyrosine at position 17 of nef is absolutely necessary for the unusual phenotype of SIVpbj14 and is sufficient to convert SIVmac239 to a virus with a phenotype like that of SIVpbjl4. Multiple NF-kappaB binding sites are not required for the in vitro properties or for acute disease.

Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication

We constructed a series of human immunodeficiency virus 1 (HIV-1)/simian immunodeficiency virus strain mac (SIVmac) chimeric viruses having vpr and/or nef genes of either HIV-1 or SIVmac based on a chimeric virus with LTRs, gag, pol, vif and vpx derived from SIVmac and tar, rev, vpu and env from HIV-1. All of the chimeric viruses replicated in human and macaque peripheral blood mononuclear cells (PBMCs) and in several CD4+ human cell lines, though their growth potentials were slightly different depending on whether vpr and nef were from HIV-1 or SIVmac, or were defective. The presence of nef accelerated replication in all the cells used and the replication of each chimera appeared to reflect that of the parental virus from which nef was derived. The presence of vpr had no clear effect in human and monkey PBMCs, but the replication of each chimera was influenced by the origin of vpr in H9 and A3.01 cells. NM-3rN, which carries HIV-1 vpr and SIVmac nef, was inoculated intravenously into three rhesus monkeys, three cynomolgus monkeys and two pig-tailed monkeys. From 2 to 14 weeks after inoculation, viruses were consistently re-isolated from all the monkeys and virus loads were as high as that of SIVmac reported previously. The results indicate that infection with NM-3rN is more efficient than any of our previous chimeric viruses and suggest that NM-3rN, having HIV-1 Env, will be a useful challenge virus for evaluating AIDS vaccines based on HIV-1 Env in macaque monkeys instead of chimpanzees.

Mutational analysis of the simian immunodeficiency virus SIVmac nef gene

We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey.