Peripheral blood mononuclear cells produce normal amounts of defective Vif- human immunodeficiency virus type 1 particles which are restricted for the preretrotranscription steps

Abstract

Previous studies have demonstrated the absence of viral replication of Vif ؊ mutants in stimulated primary blood mononuclear cells (PBMC). Human immunodeficiency virus type 1 strain NDK Vif ؊ mutants were propagated on the semipermissive CEM cell line, and the viral stock obtained was compared with the wild-type virus during a single cycle in PBMC. The Vif ؊ virus was able to enter PBMC with the same efficiency as the wild type, as demonstrated by quantification of the strong-stop cDNA, and retrotranscription was observed for both viruses within 4 h postinfection. Using a PCR assay with an Alu-long terminal repeat pair of primers, we detected integration for both the wild-type and Vif ؊ viruses. We then used qualitative and quantitative reverse transcription-mediated PCR techniques to study the steady-state level of intracellular and extracellular viral RNAs. All mRNA species were detected in PBMC infected with the wild-type virus or with the Vif ؊ virus 36 h postinfection. Furthermore, quantification of viral RNA released from infected cells demonstrated similar levels of virus produced after a unique cycle of replication. However, the Vif ؊ virus obtained after one replication cycle in PBMC was unable to initiate retrotranscription in permissive target cells. These data strongly suggest that the failure to infect target cells is due to a defect in the formation of the viral particle in PBMC.

Role of Vif in human immunodeficiency virus type 1 reverse transcription

“…The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity (10,36,38). vif mutant HIV-1 virions are defective in their ability to synthesize proviral DNA in vivo (3,7,37,40). Whether this reflects an intrinsic defect in reverse transcription or an abnormality in a preceding early event(s) such as virus entry or uncoating is unknown.…”

“…Whether this reflects an intrinsic defect in reverse transcription or an abnormality in a preceding early event(s) such as virus entry or uncoating is unknown. Peripheral blood mononuclear cells (PBMC) and some cell lines such as H9 require Vif to produce infectious HIV-1 virus particles, whereas other cell lines such as COS-1 and SupT1 do not (3,7,9,14,15,33,40). vif mutant virions produced in some nonpermissive cell lines can initiate reverse transcription but are defective in their ability to complete proviral DNA synthesis after acute infection (35,37,40).…”

Mechanistic aspects of HIV‐1 reverse transcription initiation

“…Interestingly, the degree to which these viruses are able to perform RTN after infection is determined by the permissive/restrictive phenotype of the producer cells. For example, Vif-deficient virus made in permissive cells can efficiently make proviral DNA after infection into non-permissive cells, and Vif-deficient virus made by semi-permissive CEM cells can achieve the first round of RTN and integration, but subsequent viral rounds of infection are then inhibited at RTN [31,32]. The same relationship is observed for normal and Vif-deficient virus made by either Vifpermissive or restrictive cells in endogenous RTN reactions.…”

“…HIV-1 RTN efficiency is also affected by a number of viral proteins including Nef [26,27], Tat [28][29][30] and Vif [31,32], Vpr [33], matrix protein (MA) [34][35][36], nucleocapsid protein (NCp7) [37] and integrase (IN) [38,39] (Figure 1). Nef and Vif are believed to affect virion morphology so that, in their absence, a defect in steps between entry and uncoating leads to inefficient proviral DNA synthesis.…”

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

“…There are many available assays that have been reported and used to quantify HIV DNA, of which most assays target the ltr [16,17,[19][20][21][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58], gag [22,23,43,44,54,56,[59][60][61][62], or pol [19,25,58,60,[63][64][65][66][67] regions [68]. To the best of our knowledge, we queried the target sequence of the most prominent assays to all known subtype and CRF entries in the LANL HIV1 Complete Nucleotide Filtered web alignment (http://www.hiv.lanl.gov/).…”