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

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.

The nucleocapsid region of human immunodeficiency virus type 1 Gag assists in the coordination of assembly and Gag processing: role for RNA-Gag binding in the early stages of assembly.

Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.

Mutations in human immunodeficiency virus type 1 nucleocapsid protein zinc fingers cause premature reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP((-)) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55(Gag) processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway.

The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.