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Vinay K. Pathak, Ph.D.
Viral and Host Factors That Generate Mutations in HIV-1 and MLV, Antiviral Drug Resistance, and in Vivo Reverse Transcription
1. Mechanism of APOBEC3G-mediated hypermutation and inhibition of HIV-1 replication. HIV-1 and other retroviruses occasionally undergo a high rate of G-to-A substitutions, a phenomenon named hypermutation (Pathak & Temin, PNAS, 1990). HIV-1 genomes that fail to express the accessory protein Vif cannot replicate in primary cells or 'nonpermissive' cell lines but can replicate in 'permissive' cell lines. APOBEC3G, the dominant-acting host restriction factor responsible for the nonpermissive phenotype, is a cytidine deaminase that is packaged into HIV-1 virions in the absence of Vif and deaminates deoxycytidines in minus-strand DNA to deoxyuridines, resulting in massive G-to-A hypermutation and abrogation of viral replication. HIV-1 Vif binds to APOBEC3G and induces its proteosomal degradation in the virus producer cells, suppressing its virion incorporation and restoring viral replication. Human APOBEC3G is degraded in the presence of Vif but simian APOBEC3G is resistant to Vif; using mutational analysis, we and others recently showed that a single amino acid (D128) is responsible for the species specificity of APOBEC3G proteins (Xu et al., PNAS, 2004). The D128 residue either directly interacts with Vif or is involved in conformational changes that occur upon Vif binding. To gain insights into the mechanism by which APOBEC3G inhibits HIV-1 replication, we recently developed a sensitive cytidine deamination assay using scintillation proximity beads. Using this assay, we demonstrated that interactions with viral and nonviral RNAs that are packaged are sufficient for APOBEC3G virion incorporation and that interactions with viral proteins are not essential for virion incorporation (Svarovskaia, JBC, 2004).
Our future goals are to elucidate the structure and function of APOBEC3G, identify other host proteins that are critical for APOBEC3G-mediated inhibition of HIV-1 replication, define the nature of the Vif-APOBEC3G interactions, and develop agents that interfere with Vif-APOBEC3G interactions as potential antiviral agents.
2. Mechanisms of retroviral recombination and antiviral drug resistance. Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. We have analyzed the mechanism of reverse transcriptase (RT) template-switching events in homologous repeats inserted into MLV and HIV-1 vectors. Based on these studies, we have described a novel mechanism of HIV-1 recombination referred to as dynamic copy choice (Nikolenko et al., J. Virol., 2004). The results of these studies indicate that a dynamic steady state between polymerase and RNase H activities is important for HIV-1 RT template switching. In addition, we have observed that several mutations in HIV-1 RT associated with resistance to nucleoside analogs dramatically increased RT template-switching frequencies. These results indicate that mutations conferring resistance to antiviral drugs may increase the rate of retroviral recombination and viral evolution.
Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of RT template switching, we propose that an equilibrium exists between 1) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and 2) degradation of the RNA template by RNase H activity leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3'-azido-3'-deoxythymidine (AZT) and 3-didehydro-2,3-dideoxythymidine (d4T) by as much as 180- and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers (Nikolenko et al., PNAS, 2005). These results provide novel insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in RT.
Our future goals are to analyze the role of RNase H in NRTI resistance in drug-naive and treated patients, in cell-based assays, and in biochemical studies.
3. Mechanisms of in vivo reverse transcription and development of a novel strand-specific amplification (SSA) assay. To elucidate the mechanisms of in vivo reverse transcription, we have characterized HIV-1 vectors containing two primer-binding sites and developed a quantitative in vivo assay for function of reverse transcription initiation complexes (Voronin & Pathak, J. Virol., 2004). In other studies, we have performed mutational analysis of the MLV RNase H primer grip domain to elucidate its role in fidelity of reverse transcription (Mbisa et al., J. Virol., 2005).
Conventional PCR methods cannot distinguish between the two DNA strands. We have developed a novel SSA assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in vivo (D.C. Thomas, Y.A. Voronin, G.N. Nikolenko, and V.K. Pathak, unpublished). Using SSA, we have determined for the first time the in vivo rate of HIV-1 minus-strand DNA synthesis (68 nt/min), minus-strand DNA transfer (1 min), plus-strand DNA transfer (27 min), and initiation of plus-strand DNA synthesis (8 min). The results also indicate that plus-strand DNA synthesis is initiated at multiple sites and that several RT inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their in vivo mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate development of novel antiretroviral drugs that target specific steps in HIV-1 reverse transcription.
The SSA method provides a widely applicable technology for strand-specific analysis of in vivo reverse transcription. We will use the SSA method to analyze the effects of mutations in RT, nucleocapsid, integrase, viral accessory proteins, and host restriction factors on the kinetics of in vivo reverse transcription. We will also use the SSA method to elucidate the mechanism of action of antiretroviral agents.
This page was last updated on 2/21/2013.