William J. Bosche

Human Immunodeficiency Virus Type 1 Nucleocapsid Zn2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid (NC) Zn2+ finger domains have greatly reduced infectivity, even though genome packaging is largely unaffected in certain cases. To examine replication defects, viral DNA (vDNA) was isolated from cells infected with viruses containing His-to-Cys changes in their Zn2+ fingers (NCH23C and NCH44C), an integrase mutant (IND116N), a double mutant (NCH23C/IND116N), or wild-type HIV-1. In vitro assays have established potential roles for NC in reverse transcription and integration. In vivo results for these processes were obtained by quantitative PCR, cloning of PCR products, and comparison of the quantity and composition of vDNA generated at discrete points during reverse transcription. Quantitative analysis of the reverse transcription intermediates for these species strongly suggests decreased stability of the DNA produced. Both Zn2+ finger mutants appear to be defective in DNA synthesis, with the minus- and plus-strand transfer processes being affected while interior portions of the vDNA remain more intact. Sequences obtained from PCR amplification and cloning of 2-LTR circle junction fragments revealed that the NC mutants had a phenotype similar to the IN mutant; removal of the terminal CA dinucleotides necessary for integration of the vDNA is disabled by the NC mutations. Thus, the loss of infectivity in these NC mutants in vivo appears to result from defective reverse transcription and integration processes stemming from decreased protection of the full-length vDNA. Finally, these results indicate that the chaperone activity of NC extends from the management of viral RNA through to the full-length vDNA.

The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6.

BackgroundExpression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration.ResultsThis work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection.ConclusionsThese results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.

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

ABSTRACT 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 Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

Human Cellular Nucleic Acid-Binding Protein Zn2+ Fingers Support Replication of Human Immunodeficiency Virus Type 1 When They Are Substituted in the Nucleocapsid Protein

ABSTRACT A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn2+ fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn2+ fingers. The sequence of the NH2-terminal NC Zn2+ finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn2+ finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn2+ fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn2+ finger compared to mutant and wild-type Zn2+ fingers in NC that support replication. The present study shows that six of seven of the Zn2+ fingers from the CNBP protein can be used as substitutes for the Zn2+ finger in the NH2-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn2+ finger mutants.