Mutational and functional analysis of the human immunodeficiency virus type 2 virion infectivity factor.

摘要

The human and simian immunodeficiency viruses (HIV-l, -2, and SIV) contain a well-conserved open reading frame termed the virion infectivity factor (vif) which has been implicated to play a role in viral infectivity. No studies to date have systematically examined the role of this viral gene product in HIV/SIV replication. This dissertation describes a series of molecular, immunological, and biochemical studies that were undertaken to examine directly the role of vif in the early events of viral replication and to examine its interaction with other viral proteins as it pertains to infectivity.; A series of five vif site-directed mutant isogenic proviruses were constructed and their biological and replicative properties were determined in CD4{dollar}sp-{dollar} and CD4{dollar}sp+{dollar} cell targets. All five vif mutants were significantly impaired in cell-free viral transmission ({dollar}>{dollar}100 fold) to PHA-stimulated PBL and the immortalized CD4{dollar}sp+{dollar} cell lines Sup-T1, H9, and CEMX174. Quantitative PCR analysis demonstrated that vif-deficient viruses were capable of initiating and completing reverse transcription with similar facility as compared to wild-type virus. Temporal quantitative PCR analysis demonstrated that by 48 hr postinfection there was 10-34 fold less viral DNA accumulated in cells infected by vif-deficient virus (p {dollar}<{dollar} 0.05).; A series of immunological reagents were developed to identify, characterize, and examine the interaction of vif with other viral proteins. Western analysis identified multiple forms of the vif gene product in chronically HIV-2-infected CD4{dollar}sp+{dollar} Sup-T1 lymphocytes. However, the vif gene product was not efficiently incorporated into the virion. Western and radioimmunoprecipitation analysis of sucrose cushion purified virus demonstrated that vif-deficient and wild-type virus contained equimolar quantities of virion structural proteins, including the envelope glycoproteins gp120 and gp32. Subcellular fractionation and immunofluorescence studies utilizing a transient eukaryotic expression system demonstrated that the vif gene product displays both cytoplasmic and nuclear localization. A putative nuclear localization signal, TRKQRRRDYRR, was identified in the carboxyl terminus of the vif orf. This data suggests that the HIV-2 vif gene product plays a unique role in viral replication following virion-receptor binding, cell entry, and reverse transcription.