An experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease is critical for the study of human AIDS. SIV induces an immunodeficiency syndrome in infected macaques that is remarkably similar in pathogenesis to human AIDS. The purpose of this project is to investigate host and viral factors involved in variable disease progression in SIV-infected macaques and the lack of disease in African primates infected with their own strains of SIV. ROLE OF TRIM5 ALPHA IN SIV PATHOGENESIS. To investigate the role of host factors in SIV-infection of macaques, we used a well-defined molecularly cloned virus (SIVsmE543-3), two macaque passages from the original sooty mangabey host. Our studies identified that allelic polymorphisms in the SPRY domain of TRIM5 alpha gene are responsible for much of the interindividual variation in viremia. In contrast, SIVmac appears to be adapted for resistance to rhesus macaque TRIM5. We evaluated the effect of restrictive TRIM5 alleles (TRIM-TFP and TRIM-CypA) on viremia in SIVsmE543-3-infected rhesus macaques. Restrictive genotypes were associated with significantly lower viremia than in macaques with the permissive genotype, and with emergence of escape mutations in the SIV capsid protein. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5TFP restriction. Introduction of these mutations into the original SIVsmE543-3 clone resulted in escape from TRIM5 restriction in vitro and improved virus fitness in macaques with homozygous restrictive TRIM alleles in vivo. Recent ongoing studies have shown that introduction of these two capsid mutations to the related SIVsmE660-FL14 clone also conferred escape from TRIM5 restriction. As described in the vaccine project (AI000686) we have derived E660 variants with Tier 2 neutralizing properties but these clones are still sensitive to TRIM restriction. Studies from the Watkins and Letvin labs suggest that TRIM5 polymorphisms and in particular expression of the restrictive TRIM-TFP allele contributes to protection in low dose SIVsmE660 challenge models. Therefore we have generated minimal point mutants in CA that are now resistant to TRIM restriction that are presently under evaluation in macaques with permissive or restrictive TRIM5 gentypes. These clones would be ideal source of challenge strains that should avoid TRIM restriction as a confounding factor in low dose E660 challenge models. MOLECULAR MECHANISMS OF TRIM5 RESTRICTION. In order to establish infection, immunodeficiency viruses encountered and adapted to many host restriction factors that target various steps of virus replication. In rhesus macaques, HIV-1 cores are countered by TRIM5, a host innate immunity restriction factor present in the cytoplasm of target cells. Recognition of incoming HIV-1 capsid lattice by TRIM5 results in its own multimerization into a complementary lattice, the acceleration of HIV-1 cores and the activation of TRIM5 ubiquitin ligases activity. We recently generated ubiquitination-resistant rhesus TRIM5 proteins and showed that while the recognition and destabilization by the deubiquitinated TRIM5 (DUb-TRIM5) of incoming HIV-1 cores have been preserved, reverse transcription complexes (RTC) failed to reach the nucleus and accumulated in the cytoplasm, indicating the inhibition of an early step in the HIV-1 viral life cycle that precedes provirus integration (Campbell et al, 2015, JVI). The generation of these new tools provides the unique opportunity to capture and analyze the otherwise elusive RTC in cells. We are using biochemical, functional assays in combination with imaging to isolate and examine the composition of these DUb-TRIM5 arrested RTCs. Using multiple tags and fluorescently labeled functional HIV-1 Integrase as well as capsid binding cellular protein--to visualize viral cores--we are monitoring incoming DUb-TRIM5 delayed RTCs in living cells in order to analyze their behavior as well as interaction(s) with the cell and TRIM5. These studies should help elucidate the mechanisms involved in HIV-1 provirus pre-integration events and help design strategies to inhibit RTC delivery to nucleus. NEUROAIDS. SIV and HIV are both associated with the development of encephalitis. For HIV, the onset of AIDS dementia is generally a late stage finding. In contrast, most models of SIV encephalitis (SIVE) use animals that progress rapidly to disease. We have performed sequential intravenous passage of virus isolated from the brain of rhesus macaques with SIV encephalitis and derived a viral swarm, SIVsmH804E that induces SIV meningitis and/or encephalitis at high frequencies. We assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection and observed compartmentalization of viral populations between the meninges and the parenchyma. A distinguishing feature of this neurovirulent isolate is the ability to replicate efficiently in monocyte derived macrophages (MDM). We identified substitutions in the cytoplasmic tail of the envelope gp41 protein that are associated with enhanced replication in MDM and have shown that these substitutions also enhance the ability of envelope of this virus to counteract the host restriction factor, BST-2. We have derived a molecular clone from the neurovirulent isolate, SIVsm804E-757CL that induces neuroAIDS in a high proportion of rhesus macaques with permissive TRIM5 alleles. In addition we have introduced the Gag capsid mutations identified as conferring resistance to TRIM5 inhibition into this clone and are assessing whether this new virus is capable of inducing neuroAIDS in macaques with restrictive TRIM5 genotypes. This would give us more flexibility in designing studies of neuroAIDS. Finally we are evaluating mononuclear populations isolated from the brains of macaques with and without encephalitis and using cell sorting and quantitative PCR for SIV to identify cell types infected in the brain. ASYMPTOMATIC INFECTION OF NATURAL HOST SPECIES. A second goal of this project is to study the mechanisms underlying the lack of pathogenicity of SIV for their natural host species, with emphasis on SIVagm from vervet monkeys. The maintenance of a disease-free course of SIV infection in AGM likely depends on a number of mechanisms. There are a number of distinctive features of natural infection with SIV including: 1) low numbers of CD4+ T cells, and 2) low expression of CCR5 at mucosal sites. Since AGM characteristically express very low levels of CCR5 on CD4+ T cells, it is possible that SIVagm this may be a common theme among natural hosts species. Initial studies using the CCR5 inhibitor, Maraviroc did not block infection indicating the use of alternative coreceptors in this virus model. We examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4(+) T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4(+) T cells and are potential alternative coreceptors for SIVagm use in vivo.

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34
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2016
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Vinton, Carol L; Ortiz, Alexandra M; Calantone, Nina et al. (2017) Cytotoxic T Cell Functions Accumulate When CD4 Is Downregulated by CD4+ T Cells in African Green Monkeys. J Immunol 198:4403-4412
Matsuda, Kenta; Riddick, Nadeene E; Lee, Cheri A et al. (2017) A SIV molecular clone that targets the CNS and induces neuroAIDS in rhesus macaques. PLoS Pathog 13:e1006538
Riddick, Nadeene E; Wu, Fan; Matsuda, Kenta et al. (2016) Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors. J Virol 90:2316-31
Hessell, Ann J; Jaworski, J Pablo; Epson, Erin et al. (2016) Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques. Nat Med 22:362-8
DiNapoli, Sarah R; Hirsch, Vanessa M; Brenchley, Jason M (2016) Macrophages in Progressive Human Immunodeficiency Virus/Simian Immunodeficiency Virus Infections. J Virol 90:7596-606
Mudd, Joseph C; Perkins, Molly R; DiNapoli, Sarah R et al. (2016) Interleukin-2 Therapy Induces CD4 Downregulation, Which Decreases Circulating CD4 T Cell Counts, in African Green Monkeys. J Virol 90:5750-8
Matsuda, Kenta; Chen, Chia-Yen; Whitted, Sonya et al. (2016) Enhanced antagonism of BST-2 by a neurovirulent SIV envelope. J Clin Invest 126:2295-307
Vinton, Carol; Wu, Fan; Rossjohn, Jamie et al. (2016) Mucosa-Associated Invariant T Cells Are Systemically Depleted in Simian Immunodeficiency Virus-Infected Rhesus Macaques. J Virol 90:4520-9
Takeuchi, Junko S; Ren, Fengrong; Yoshikawa, Rokusuke et al. (2015) Coevolutionary dynamics between tribe Cercopithecini tetherins and their lentiviruses. Sci Rep 5:16021
Campbell, Edward M; Weingart, Jared; Sette, Paola et al. (2015) TRIM5?-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization. J Virol 90:1849-57

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