Determining the Functional Significance of Mutations Observed in Envelope Protein Following Serial In Vivo Passaging of Human-Simian Immunodeficiency Virus

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Abstract/Summary: Commonly used animal models of HIV-1 include infection of macaques with Simian Immunodeficiency Virus (SIV) or Simian-Human Immunodeficiency Virus (SHIV) containing HIV envelope (Env) or reverse transcriptase. These animal models have been extremely useful in understanding HIV pathogenesis and disease progression, as well as understanding the efficacy of vaccines and drugs. However, the genetic difference between HIV-1 and SIV, and the absence of other HIV-1 genes such as gag, pol, vif, vpr, and nef in SHIV limits the utility of these models in vaccine studies. Ideally, good animal model of HIV-1 infection/AIDS would be infection of macaques with HIV-1. However, HIV-1 does not replicate in macaque cells due to the presence of retroviral restriction factors. HIV-1 can be made to replicate by substituting its accessory genes with SIV genes such as vif, vpx, vpr, and nef, which can counteract interferon-induced restriction factors in macaque cells. Human-Simian Immunodeficiency Virus (HSIV) is an HIV-1NL4-3 derivative with SIV vif gene substitution (named HSIV-vifNL4-3) that can replicate persistently in pigtail macaques (PTMs). However, infection did not result in high peak viremia and setpoint viral loads as observed during SIV infection of macaques. Serial in vivo passaging in PTMs was performed to enhance infectivity or replicative capacity of HSIV. Three rounds of animal-to-animal transfer of infected blood in 3 immunocompetent PTMs with starting initial inoculum containing a mixture of CXCR4- (HSIV- vifNL4-3 recovered from previously infected macaque) and CCR5-tropic HSIV (HSIV-vif derivative based on pNL- AD8 and Bru-Yu2) was conducted to generate pathogenic variants. Interestingly, all the macaques showed peak viremia close to or above 105 copies/ml and virus replication persisted for more than 20 weeks. Following in vivo passaging, three infectious molecular clones (IMCs) were recovered from passage 3 macaque (HSIV-P3 IMCs). Sequencing of HSIV-P3 IMCs showed several interesting mutations throughout the genome, perhaps suggesting adaptation to PTMs. These mutations could help the virus in overcoming restriction factors, or better utilization of host dependency factors, or they could help the virus escape host immune responses. Focus of this grant application is to determine the functional significance of mutations observed in envelope gene. The results from this study will provide valuable insights into the role of envelope gene in cross-species transmission of HIV-1 to pigtailed macaques.