Jianhui Guo

Nucleic Acid Chaperone Activity of HIV‐1 Nucleocapsid Protein: Critical Role in Reverse Transcription and Molecular Mechanism

Publisher Summary This chapter focuses on recent biochemical and biophysical studies that examine the nucleic acid chaperone function of HIV‐1 nucleocapsid protein (NC) and its critical role in facilitating specific and efficient reverse transcription. This chapter also describes the effect of NC on individual steps in viral DNA synthesis. This chapter also summarizes what is known about NC structure, NC nucleic acid binding properties, and the contribution of the zinc fingers to chaperone activity. In addition, this chapter also discusses new evidence that provides a model to explain the mechanism of NCs nucleic acid chaperone activity at the molecular level. Characterization of the mechanism of NCs chaperone activity in molecular terms has been invaluable for understanding NCs effect on specific steps in reverse transcription. NC binds nucleic acids noncooperatively and does not rely on protein– protein interactions to drive aggregation and annealing. Instead, NC‐induced nucleic acid aggregation appears to be facilitated by simple polyelectrolyte attraction, similar to that observed for many multivalent cations.

Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein

ABSTRACT The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus- and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.

Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3′ polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5′-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NCs observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Actinomycin D Induces High-Level Resistance to Thymidine Analogs in Replication of Human Immunodeficiency Virus Type 1 by Interfering with Host Cell Thymidine Kinase Expression

ABSTRACT Actinomycin D (ActD) is a transcription inhibitor and has been used in the treatment of certain forms of cancer. ActD has been reported to be a potential inhibitor of human immunodeficiency virus type 1 (HIV-1) replication due to its ability to inhibit reverse transcription. In contrast to what was expected, low concentrations of ActD (1 to 10 nM) upregulated HIV-1 replication 8- to 10-fold in MT-2 cells and had no effect on HIV-2 replication or on HIV-1 replication in MT-4, Jurkat, or peripheral blood mononuclear cells. The upregulation of HIV-1 replication was associated with an increase in HIV-1 transcription and a decrease in CD4 and CXCR4 expression. To further evaluate the effects of ActD on emergence of drug resistance in HIV-1 replication, a series of drug resistance assays were performed. Of interest, treatment of MT-2 cells with ActD also led to a high level of resistance to thymidine analogs (>1,000-fold increase in resistance to zidovudine and >250-fold to stavudine) but not to other nucleoside reverse transcriptases (RT), nonnucleoside RT, or protease inhibitors. This resistance appeared to be due to a suppression of host cell thymidine kinase-1 (TK-1) expression. These results indicate that ActD leads to a novel form of thymidine analog resistance by suppressing host cell TK-1 expression. These results suggest that administration of combination drugs to HIV-1-infected patients may induce resistance to antiretroviral compounds via a modification of cellular factors.

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.