Abstract
BackgroundThe machinery of early HIV-1 replication still remains to be elucidated. Recently the viral core was reported to persist in the infected cell cytoplasm as an assembled particle, giving rise to the reverse transcription complex responsible for the synthesis of proviral DNA and its transport to the nucleus. Numerous studies have demonstrated that reverse transcription of the HIV-1 genome into proviral DNA is tightly dependent upon proper assembly of the capsid (CA) protein into mature cores that display appropriate stability. The functional impact of structural properties of the core in early replicative steps has yet to be determined.ResultsHere, we show that infectivity of HIV-1 mutants bearing S149A and S178A mutations in CA can be efficiently restored when pseudotyped with vesicular stomatitis virus envelope glycoprotein, that addresses the mutant cores through the endocytic pathway rather than by fusion at the plasma membrane. The mechanisms by which these mutations disrupt virus infectivity were investigated. S149A and S178A mutants were unable to complete reverse transcription and/or produce 2-LTR DNA. Morphological analysis of viral particles and in vitro uncoating assays of isolated cores demonstrated that infectivity defects resulted from disruption of the viral core assembly and stability for S149A and S178A mutants, respectively. Consistent with these results, both mutants failed to saturate TRIM-antiviral restriction activity.ConclusionDefects generated at the level of core assembly and stability by S149A and S178A mutations are sensitive to the way of delivery of viral nucleoprotein complexes into the target cell. Addressing CA mutants through the endocytic pathway may compensate for defects generated at the reverse transcription/nuclear import level subsequent to impairment of core assembly or stability.
Highlights
The genome of the human immunodeficiency virus type 1 (HIV-1) is packaged within a conical shaped core formed by the viral capsid protein (CA) and delivered to the host cell cytoplasm upon fusion of the viral and cell membranes
All three mutants were found to be poorly infectious compared to the NL4.3 wild-type (WT) viruses when viral input was normalized according to reverse transcriptase (RTase) activity
Pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) significantly restored infectious properties of S149A and S178A mutants to levels observed for VSV-G-WT viruses
Summary
The genome of the human immunodeficiency virus type 1 (HIV-1) is packaged within a conical shaped core formed by the viral capsid protein (CA) and delivered to the host cell cytoplasm upon fusion of the viral and cell membranes. HIV-1 cores may not dissociate immediately after the viral fusion, but rather remain largely intact for at least a portion of the process from the initiation of RT to the synthesis of the central flap structure [7,8] This model is further supported by the ability of RT to progress efficiently in intact virions, allowing the synthesis of fulllength minus strand DNA in this core fraction, without requirement for an uncoating activity [2]. Drugs altering the integrity of the cytoskeleton [12] and RNA interference targeting the actin nucleator Arp2/3 complex [13] inhibit post-entry steps of the retroviral cycle These data agree with imaging analysis in living cells showing that fluorescent HIV-1 complexes migrate as assembled cores along the actin cytoskeleton and microtubule network before being addressed to the microtubule-organizing center in the perinuclear region [6] or even to the nuclear pore itself [7] where uncoating may take place. The functional impact of structural properties of the core in early replicative steps has yet to be determined
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