Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3GCTD and in that of full-length wild-type APOBEC3G.

Genetic analysis of the localization of APOBEC3F to human immunodeficiency virus type 1 virion cores.

UNLABELLED: Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287:16965-16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization. IMPORTANCE: Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

Signals in APOBEC3F N-terminal and C-terminal deaminase domains each contribute to encapsidation in HIV-1 virions and are both required for HIV-1 restriction.

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) inhibit human immunodeficiency virus type-1 (HIV-1) replication. In the absence of HIV-1 Vif, A3F and/or A3G are incorporated into assembling virions and exert antiviral functions in subsequently infected target cells. Encapsidation of A3F or A3G within the protease-matured virion core following their incorporation into virions is hypothesized to be important for the antiviral function of these proteins. In this report, we demonstrated that A3F was quantitatively encapsidated in the mature virion core. In distinct contrast, A3G was distributed both within and outside of the virion core. Analysis of a series of A3F-A3G chimeras comprised of exchanged N- and C-terminal deaminase domains identified a 14 amino acid segment in the A3F C-terminal deaminase domain that contributed to preferential encapsidation and anti-HIV activity. Amino acid residue L306 in this C-terminal segment was determined to be necessary, but not sufficient, for these effects. Amino acid residue W126 in the N-terminal deaminase domain was determined also to contribute to preferential encapsidation and antiviral activity of A3F. Analysis of the A3F (W126A L306A) double mutant revealed that both residues are required for full anti-HIV function. The results reported here advance our understanding of the mechanisms of A3F virion encapsidation and antiviral function and may lead to innovative strategies to inhibit HIV-1 replication.

Differences in HIV-1 pol sequences from female genital tract and blood during antiretroviral therapy.

OBJECTIVE: To determine whether HIV-1 replicates locally in the female genital tract during therapy, and to study whether endocervix is the dominant source of virus in cervicovaginal lavage fluid. DESIGN: Sequence analyses of HIV-1 pol were performed from cervicovaginal secretions and blood plasma of HIV-infected women failing antiretroviral therapy with detectable viral load in both compartments, as well as from drug-naive subjects. METHODS: Viral RNA was extracted from cervicovaginal lavage fluid, endocervical secretions collected by Sno-strips, and blood plasma. Population sequencing of HIV-1 pol was performed using cycle sequencing. Drug resistance mutations were analyzed. Phylogenies were constructed based on synonymous positions in the sequences. RESULTS: Resistant virus was detected concordantly in blood and genital tract specimens, consistent with drug selection pressure in both compartments. However, drug-selected mutations often differed in each compartment, and phylogenetic analysis showed differences in virus lineage in these compartments, consistent with local replication in female genital tract. Viruses in cervicovaginal lavage and endocervical secretions were genetically distinguishable, suggesting that endocervix is not the only source of virus found in cervicovaginal lavage. CONCLUSION: These data support the hypothesis that HIV replication is compartmentalized within the female genital tract during antiretroviral therapy, which has implications for pathogenesis and for epidemiologic surveillance of drug-resistant virus.

Antiretroviral resistance associated with supervised treatment interruptions in treated acute HIV infection.

We studied 14 patients acutely infected with wild-type HIV, who underwent supervised treatment interruptions after initial antiretroviral treatment including lamivudine. Lamivudine resistance mutations emerged for the first time during supervised treatment interruptions in one patient. Resistance should be monitored in supervised treatment interruptions trials, because mutations may first be detected only after therapy is interrupted.

Antiretroviral drug resistance mutations in antiretroviral-naive prisoners.

We assessed the incidence of antiretroviral drug resistance in a cohort of 25 antiretroviral-naive, human immunodeficiency virus-positive inmates in Massachusetts. Silent mutations, unexpected mutations at resistant sites, and resistance mutations were recorded. Among these inmates, we found a prevalence of drug resistance mutations that was equivalent to the prevalence previously found in nonprison populations in the same state.