12th Conference on Retroviruses and Opportunistic Infections Boston, Massachusetts - February 22-25, 2005

Conf Retrovir Opportunistic Infect 2005 Feb 22-25;12:abstract no. 29

Bärbel Schröfelbauer, Q Yu, and N Landau
Salk Inst for Biological Studies, San Diego, CA, USA

BACKGROUND: APOBEC3 family cytidine deaminases are potent inhibitors of lentiviral replication. The HIV-1 accessory protein, Vif, induces the degradation of the deaminases that are otherwise encapsidated and subsequently catalyze C→U deamination of the viral reverse transcript. DNA repair enzymes uracil DNA glycosylases (UDG) are believed to target the hypermutated transcripts resulting in their degradation. The realization that C→U deamination is a critical process in HIV-1 replication led us to reconsider the importance of the previously reported interaction between Vpr and UNG. We hypothesized that this interaction might serve as a secondary viral mechanism for further reducing damage to the viral genome caused by cellular cytidine deaminases.

METHODS: The amount of encapsidated UNG2 and SMUG1 in pelleted virions was analyzed by immunoblot. Complex formation between Vpr and UNG2, SMUG1, and Cullins was tested by co-immunoprecipitation and effects on half-life were determined by pulse-chase. Luciferase-based single-round infectivity assays were used to investigate the effect of Vpr on viral infectivity. The stability of reverse transcripts was determined by quantitative PCR. Reverse transcripts were amplified and sequenced to determine the G→A mutational frequency.

RESULTS: Vpr-deficient virions contained large amounts of the single-strand specific uracil DNA glycosylases UNG and SMUG. In contrast, Vpr+ virions contained almost none. In the presence of Vpr, UNG and SMUG were rapidly polyubiquitinated and degraded through a proteasomal pathway. Vpr formed a complex with the E3 ubiquitin ligase components, Cullin1 and Cullin4a that was readily detectable by co-immunoprecipitation. Vpr restored the infectivity of HIV-1 that had been produced in the presence of small amounts of APOBEC3G and stabilized the reverse transcripts. Vpr, with a mutation at amino acid 54 that blocks the interaction with UNG, was inactive.

CONCLUSIONS: We conclude that Vpr serves as a secondary system by which the virus further protects itself from deamination, thereby increasing the fidelity with which its genome is replicated. The presence of 2 accessory genes in the viral genome dedicated to prevent the encapsidation of cellular DNA-modifying enzymes highlights the importance of cytidine deamination in the viral life cycle.

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