Abstract
BackgroundThe nucleocapsid (NC) protein of HIV-1 is critical for viral replication. Mutational analyses have demonstrated its involvement in viral assembly, genome packaging, budding, maturation, reverse transcription, and integration. We previously reported that two conservative NC mutations, His23Cys and His44Cys, cause premature reverse transcription such that mutant virions contain approximately 1,000-fold more DNA than wild-type virus, and are replication defective. In addition, both mutants show a specific defect in integration after infection.ResultsIn the present study we investigated whether blocking premature reverse transcription would relieve the infectivity defects, which we successfully performed by transfecting proviral plasmids into cells cultured in the presence of high levels of reverse transcriptase inhibitors. After subsequent removal of the inhibitors, the resulting viruses showed no significant difference in single-round infective titer compared to viruses where premature reverse transcription did occur; there was no rescue of the infectivity defects in the NC mutants upon reverse transcriptase inhibitor treatment. Surprisingly, time-course endogenous reverse transcription assays demonstrated that the kinetics for both the NC mutants were essentially identical to wild-type when premature reverse transcription was blocked. In contrast, after infection of CD4+ HeLa cells, it was observed that while the prevention of premature reverse transcription in the NC mutants resulted in lower quantities of initial reverse transcripts, the kinetics of reverse transcription were not restored to that of untreated wild-type HIV-1.ConclusionsPremature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.
Highlights
The nucleocapsid (NC) protein of HIV-1 is critical for viral replication
When we examined the nucleic acids present in NC-mutant virions prior to infection, we found that they contained a significant amount of viral DNA (vDNA) (~1,000-fold more than WT [33]); virtually every particle had initiated reverse transcription, and so this process is apparently occurring prematurely in the viral replication cycle
Reverse transcriptase inhibitors prevent infection and can be effectively removed from virus preparations Initial experiments were performed to determine the necessary concentrations of reverse transcriptase inhibitors (RTIs) to use and we found that a single inhibitor was insufficient to block the levels of premature reverse transcription that the NC mutations were causing
Summary
Mutational analyses have demonstrated its involvement in viral assembly, genome packaging, budding, maturation, reverse transcription, and integration. We previously reported that two conservative NC mutations, His23Cys and His44Cys, cause premature reverse transcription such that mutant virions contain approximately 1,000-fold more DNA than wild-type virus, and are replication defective. Both mutants show a specific defect in integration after infection. The nucleocapsid (NC) protein of HIV-1 functions throughout the viral replication cycle, from involvement in assembly and genomic RNA (gRNA) packaging as part of the Gag protein (Pr55), to facilitating reverse transcription as a mature protein (p7). The general properties of NC as a nucleic acid chaperone were observed many years ago in vitro [17,27], the mechanics of how these properties govern NC’s actions during reverse transcription is still being elucidated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
