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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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NC mutant reverse transcription kinetics in cells are altered when premature reverse transcription is blocked. CD4+ HeLa cells were infected with virus prepared in the absence or presence of RTIs that were subsequently removed using PEG-precipitation (Figure 1, left). These charts display the profile of reverse transcripts over a 72 h time course of infection. Panels A, C, and E show infections from viruses (WT, NCH23C, and NCH44C, respectively) not treated with RTIs, and panels B, D, and F show infections from viruses (WT, NCH23C, and NCH44C, respectively) where premature reverse transcription was blocked via RTI treatment. Prior to the infection, all of the virus samples were normalized for RT activity so that equal amounts were used to infect each set of cells. These results are from a representative experiment. The vDNA species measured were normalized for cell equivalents using CCR5 and are indicated at the bottom of panel A. Schematics of the pertinent vDNA target sites are shown at the bottom of Figure 3.
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Figure 6: NC mutant reverse transcription kinetics in cells are altered when premature reverse transcription is blocked. CD4+ HeLa cells were infected with virus prepared in the absence or presence of RTIs that were subsequently removed using PEG-precipitation (Figure 1, left). These charts display the profile of reverse transcripts over a 72 h time course of infection. Panels A, C, and E show infections from viruses (WT, NCH23C, and NCH44C, respectively) not treated with RTIs, and panels B, D, and F show infections from viruses (WT, NCH23C, and NCH44C, respectively) where premature reverse transcription was blocked via RTI treatment. Prior to the infection, all of the virus samples were normalized for RT activity so that equal amounts were used to infect each set of cells. These results are from a representative experiment. The vDNA species measured were normalized for cell equivalents using CCR5 and are indicated at the bottom of panel A. Schematics of the pertinent vDNA target sites are shown at the bottom of Figure 3.

Mentions: The kinetic profiles of vDNA synthesis during a wild-type infection, with virus prepared without RTIs are shown in Figure 6A. This chart is similar to what we had reported previously--a maximum accumulation of vDNA occurred at 12 h post infection and by 24 h post infection the amounts of R-U5 and U3-U5 are about twice those of Gag and R-5'UTR. In addition, we do not see any evidence for reinfection, although it should be theoretically possible (the cells are CD4+ and the proviral clones are Env(+)). However, because Env, Nef, and Vpu can down regulate the CD4 receptor in infected cells [47,48], the lack of reinfection is not necessarily surprising. Figures 6C and 6E show the vDNA profiles after infection with the NCH23C and NCH44C mutants (without RTI treatment), respectively. As we previously reported, quantities of vDNA at 4 h were similar to wild-type, but unlike wild-type, these were the maximum levels achieved during the entire time course of infection [32].


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

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

NC mutant reverse transcription kinetics in cells are altered when premature reverse transcription is blocked. CD4+ HeLa cells were infected with virus prepared in the absence or presence of RTIs that were subsequently removed using PEG-precipitation (Figure 1, left). These charts display the profile of reverse transcripts over a 72 h time course of infection. Panels A, C, and E show infections from viruses (WT, NCH23C, and NCH44C, respectively) not treated with RTIs, and panels B, D, and F show infections from viruses (WT, NCH23C, and NCH44C, respectively) where premature reverse transcription was blocked via RTI treatment. Prior to the infection, all of the virus samples were normalized for RT activity so that equal amounts were used to infect each set of cells. These results are from a representative experiment. The vDNA species measured were normalized for cell equivalents using CCR5 and are indicated at the bottom of panel A. Schematics of the pertinent vDNA target sites are shown at the bottom of Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: NC mutant reverse transcription kinetics in cells are altered when premature reverse transcription is blocked. CD4+ HeLa cells were infected with virus prepared in the absence or presence of RTIs that were subsequently removed using PEG-precipitation (Figure 1, left). These charts display the profile of reverse transcripts over a 72 h time course of infection. Panels A, C, and E show infections from viruses (WT, NCH23C, and NCH44C, respectively) not treated with RTIs, and panels B, D, and F show infections from viruses (WT, NCH23C, and NCH44C, respectively) where premature reverse transcription was blocked via RTI treatment. Prior to the infection, all of the virus samples were normalized for RT activity so that equal amounts were used to infect each set of cells. These results are from a representative experiment. The vDNA species measured were normalized for cell equivalents using CCR5 and are indicated at the bottom of panel A. Schematics of the pertinent vDNA target sites are shown at the bottom of Figure 3.
Mentions: The kinetic profiles of vDNA synthesis during a wild-type infection, with virus prepared without RTIs are shown in Figure 6A. This chart is similar to what we had reported previously--a maximum accumulation of vDNA occurred at 12 h post infection and by 24 h post infection the amounts of R-U5 and U3-U5 are about twice those of Gag and R-5'UTR. In addition, we do not see any evidence for reinfection, although it should be theoretically possible (the cells are CD4+ and the proviral clones are Env(+)). However, because Env, Nef, and Vpu can down regulate the CD4 receptor in infected cells [47,48], the lack of reinfection is not necessarily surprising. Figures 6C and 6E show the vDNA profiles after infection with the NCH23C and NCH44C mutants (without RTI treatment), respectively. As we previously reported, quantities of vDNA at 4 h were similar to wild-type, but unlike wild-type, these were the maximum levels achieved during the entire time course of infection [32].

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