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 both in terms of pathogenesis and in the development of effective vaccine strategies for 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. Allelic polymorphisms in the SPRY domain of TRIM5 alpha gene are responsible for much of the inter-individual variation in viremia with this virus. In contrast, SIVmac appears to be adapted for resistance to rhesus macaque TRIM5. Following SIVsmE543-infection, restrictive genotypes of TRIM 5 (TRIM-TFP and TRIM-CypA) 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 TRIM5-TFP 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. Introduction of these two capsid mutations to the related SIVsmE660-FL14 clone also conferred escape from TRIM5 restriction demonstrating common escape pathways from TRIM restriction in related SIV strains. MOLECULAR AND BIOCHEMICAL STUDIES OF TRIM5 FUNCTION IN HIV/SIV AND FLAVIVIRUSES. During the cellular response to viral infection, the innate immune system is triggered to recognize and respond to foreign viral proteins and viral nucleic acid-protein complexes known as RNPs. One example of this is the recognition of the Human Immunodeficiency Virus-1 (HIV-1) retroviral core entering the cytoplasm by the innate immunity protein known as TRIM5. In this context, TRIM5 is recruited to and believed to disrupt the RNP-containing incoming viral capsid cores soon after entrance into infected cells, although the precise molecular mechanism underlying this interaction remains to be determined. Recently, in collaboration with Sonja Best's laboratory, we determined that the recruitment of TRIM5 and viral restriction also occurs during the infection with flaviviruses belonging to the Tick-Borne serogroup like Tick-Borne Encephalitis Virus (TBEV). TRIM5 was recruited to flavivirus replication complexes and RNPs in the Endoplasmic Reticulum (ER) of infected cells and signaled the viral protein for degradation via the Ubiquitin-Proteasome System (UPS) in a K48-Ubiquitin-dependent manner. Now, we are interested in understanding the mechanisms underlying TRIM5 recognition of RNPs and recruitment to the ER, potentially extending these newly characterized mechanisms of TRIM5 to HIV-1 restriction. 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 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 and is distinguished by the ability to replicate efficiently in monocyte derived macrophages (MDM). Substitutions in the cytoplasmic tail of the envelope gp41 protein were found to be associated with both enhanced replication in MDM and enhanced ability 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. We 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. Finally, we evaluated 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. This study revealed SIV DNA within CD4+ T lymphocytes of the brain in all SIV-infected animals but only within brain macrophages in animals with SIV encephalitis. Future studies will use these techniques to evaluate the cellular reservoir in the brain of macaques on highly effective antiviral therapy. 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. VACCINE STUDIES: Our prior vaccine studies focused on the use of the highly attenuated modified vaccinia virus Ankara to express SIV proteins. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. Better preservation of memory CD4+ T cells was correlated with titers of neutralizing antibodies to the challenge virus prior to challenge suggesting an important role for NAb and Envelope immunogens in this model but are not currently under active study since the use of SHIV models are more appropriate. NEUTRALIZING ANTIBODY RESPONSES AFTER SIV-INFECTION: Another focus of the lab has been the characterization of neutralizing antibody responses after challenge with SIVsm. We generated full length infectious clones of the challenge stock, SIVsmE660. Two of these viruses replicate efficiently in rhesus PBMC in vitro and were very sensitive to neutralizing antibody (equivalent to Tier 1 classification used for HIV-1) and when evaluated for in vivo viral replication in rhesus macaques, resulted in robust and persistent viremia. Evolution of the V1 and V4 regions was associated with escape from neutralization. We defined at least three groups roughly equivalent to the Tiers 1, 2 and 3 and created and characterized representative Tier 1, Tier2 and Tier 3 variants from this panel. The three viruses had similar replicative fitness in rhesus macaques, as indicated by similar virus acquisition rate and plasma viremia during the acute phase of infection. A similar stepwise development of NAb responses was observed in all animals, with Tier 1 viruses neutralized early, Tier 2 viruses somewhat later and Tier 3 viruses much later after infection, regardless of which strain the macaques received. As a result, the selective pressure exerted by the NAbs against autologous viruses in the three cohorts differed, resulting in different viral divergence rates in the three groups as determined by NGS sequencing, with the most rapid divergence rate observed in the cohort infected with the Tier 1A virus. Studies are underway to evaluate the replicative fitness of these three variants in co-inoculation studies.
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