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Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect.

Thomas JA, Shatzer TL, Gorelick RJ - Retrovirology (2011)

Bottom Line: In 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.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.Premature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.

View Article: PubMed Central - HTML - PubMed

Affiliation: AIDS and Cancer Virus Program, SAIC-Frederick, Inc,, NCI at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: The 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.

Results: In 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.

Conclusions: Premature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.

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Related in: MedlinePlus

Methods to remove RTIs from virus preparations. Schematic of the two methods used to remove RTIs from virus preparations. The RTIs were removed so that they did not inhibit downstream assays to assess viral function when premature reverse transcription was blocked. In both methods aspiration was used to remove the supernatant after centrifugation (see the Methods section for details). The method on the left was used to i) maintain competent Env proteins on the surface of virions and ii) limit mechanical stress on virions for subsequent infection analyses. The method on the right uses DNase I treatment to remove extra-virion plasmid DNA contamination with subsequent subtilisin digestion to ensure that the DNase I is completely removed prior to lysis of the virions, and qPCR analysis of intravirion DNA and endogenous reverse transcription assays [33].
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Figure 1: Methods to remove RTIs from virus preparations. Schematic of the two methods used to remove RTIs from virus preparations. The RTIs were removed so that they did not inhibit downstream assays to assess viral function when premature reverse transcription was blocked. In both methods aspiration was used to remove the supernatant after centrifugation (see the Methods section for details). The method on the left was used to i) maintain competent Env proteins on the surface of virions and ii) limit mechanical stress on virions for subsequent infection analyses. The method on the right uses DNase I treatment to remove extra-virion plasmid DNA contamination with subsequent subtilisin digestion to ensure that the DNase I is completely removed prior to lysis of the virions, and qPCR analysis of intravirion DNA and endogenous reverse transcription assays [33].

Mentions: However, our investigations required determining the properties of virions after premature reverse transcription had been blocked, so we developed two different methods (Figure 1) to remove excess RTIs from virus preparations once particles were released from the producer cells and premature reverse transcription could no longer occur. Key to both of these methods is the collection of the virus particles for complete media replacement, which reduces the concentration of RTIs to levels far below what would interfere with reverse transcription. For subsequent infectivity experiments, we precipitated virus from culture supernatants with polyethylene glycol (PEG 8000) at 4°C (Figure 1, left). In contrast, for subsequent assessment of intravirion DNA levels and endogenous reverse transcription assays, we used our previously reported protocol for preparing virions (Figure 1, right; [33]); this rigorous protocol was found to be essential for removal of extra-virion contaminating plasmid DNA to enable accurate determinations of intravirion DNA levels [33]. However, virus treated by the latter method, which entails subtilisin digestion to inactivate the DNase I prior to lysing the virions cannot be used for infectivity assays as all membrane surface proteins, including Env, are digested [40].


Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect.

Thomas JA, Shatzer TL, Gorelick RJ - Retrovirology (2011)

Methods to remove RTIs from virus preparations. Schematic of the two methods used to remove RTIs from virus preparations. The RTIs were removed so that they did not inhibit downstream assays to assess viral function when premature reverse transcription was blocked. In both methods aspiration was used to remove the supernatant after centrifugation (see the Methods section for details). The method on the left was used to i) maintain competent Env proteins on the surface of virions and ii) limit mechanical stress on virions for subsequent infection analyses. The method on the right uses DNase I treatment to remove extra-virion plasmid DNA contamination with subsequent subtilisin digestion to ensure that the DNase I is completely removed prior to lysis of the virions, and qPCR analysis of intravirion DNA and endogenous reverse transcription assays [33].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141651&req=5

Figure 1: Methods to remove RTIs from virus preparations. Schematic of the two methods used to remove RTIs from virus preparations. The RTIs were removed so that they did not inhibit downstream assays to assess viral function when premature reverse transcription was blocked. In both methods aspiration was used to remove the supernatant after centrifugation (see the Methods section for details). The method on the left was used to i) maintain competent Env proteins on the surface of virions and ii) limit mechanical stress on virions for subsequent infection analyses. The method on the right uses DNase I treatment to remove extra-virion plasmid DNA contamination with subsequent subtilisin digestion to ensure that the DNase I is completely removed prior to lysis of the virions, and qPCR analysis of intravirion DNA and endogenous reverse transcription assays [33].
Mentions: However, our investigations required determining the properties of virions after premature reverse transcription had been blocked, so we developed two different methods (Figure 1) to remove excess RTIs from virus preparations once particles were released from the producer cells and premature reverse transcription could no longer occur. Key to both of these methods is the collection of the virus particles for complete media replacement, which reduces the concentration of RTIs to levels far below what would interfere with reverse transcription. For subsequent infectivity experiments, we precipitated virus from culture supernatants with polyethylene glycol (PEG 8000) at 4°C (Figure 1, left). In contrast, for subsequent assessment of intravirion DNA levels and endogenous reverse transcription assays, we used our previously reported protocol for preparing virions (Figure 1, right; [33]); this rigorous protocol was found to be essential for removal of extra-virion contaminating plasmid DNA to enable accurate determinations of intravirion DNA levels [33]. However, virus treated by the latter method, which entails subtilisin digestion to inactivate the DNase I prior to lysing the virions cannot be used for infectivity assays as all membrane surface proteins, including Env, are digested [40].

Bottom Line: In 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.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.Premature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.

View Article: PubMed Central - HTML - PubMed

Affiliation: AIDS and Cancer Virus Program, SAIC-Frederick, Inc,, NCI at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: The 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.

Results: In 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.

Conclusions: Premature reverse transcription is not the cause of the replication defect but is an independent side-effect of the NC mutations.

Show MeSH
Related in: MedlinePlus