Retrovirology - Latest Articles

Evolution of SIV toward RANTES resistance in macaques rapidly progressing to AIDS upon coinfection with HHV-6A

Angelique Biancotto — 2009-07-02T00:00:00Z

Background: Progression to AIDS is often associated with an evolution of HIV-1 toward increased virulence and/or pathogenicity. Evidence suggests that a virulence factor for HIV-1 is resistance to CCR5-binding chemokines, most notably RANTES, which are believed to play a role in HIV-1 control in vivo. HIV-1 can achieve RANTES resistance either by phenotypic switching from exclusive CCR5 usage to an expanded coreceptor specificity, or by the acquisition of alternative modalities of CCR5 usage. An infectious agent that might promote the evolution of HIV-1 toward RANTES resistance is human herpesvirus 6A (HHV-6A), which is frequently reactivated in HIV-1-infected patients and is a potent RANTES inducer in lymphoid tissue. Results: SIV isolates obtained from pig-tailed macaques (M. nemestrina) after approximately one year of single infection with SIVsmE660 or dual infection with SIVsmE660 and HHV-6AGS were characterized for their growth capacity and sensitivity to HHV-6A- and RANTES-mediated inhibition in human or macaque lymphoid tissues ex vivo. Four out of 4 HHV-6A-coinfected macaques, all of which progressed to full-blown AIDS within 2 years of infection, were found to harbor SIV variants with a reduced sensitivity to both HHV-6A and RANTES, despite maintaining an exclusive CCR5 coreceptor specificity; viruses derived from two of these animals replicated even more vigorously in the presence of exogenous HHV-6A or RANTES. The SIV variants that emerged in HHV-6A-coinfected macaques showed an overall reduced ex vivo replication capacity that was partially reversed upon addition of exogenous RANTES, associated with suppressed IL-2 and enhanced IFN-g production. In contrast, SIV isolates obtained from two singly-infected macaques, none of which progressed to AIDS, maintained HHV-6A/RANTES sensitivity, whereas the only AIDS progressor among singly-infected macaques developed an SIV variant with increased replication capacity and expanded coreceptor usage associated with partial HHV-6A/RANTES resistance. Conclusions: These results provide in vivo evidence of SIV evolution toward RANTES resistance in macaques rapidly progressing to AIDS. RANTES resistance may represent a common virulence factor allowing primate immunodeficiency retroviruses to evade a critical mechanism of host antiviral defense.

Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors

Barbara Falkensammer — 2009-06-21T00:00:00Z

Background: We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens. Results: Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p<0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres. Conclusions: The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.

OVEX1, a novel chicken endogenous retrovirus with sex-specific and left-right asymmetrical expression in gonads

Daniele Carre-Eusebe — 2009-06-17T00:00:00Z

Background: In chickens, as in most birds, female gonad morphogenesis is asymmetrical. Gonads appear first rather similarly, but only the left one undergoes full differentiation and gives rise to a functional ovary. The right gonad, in which the cortex does not develop, remains restricted to the medulla and finally regresses. Opportunity was taken of this left-right asymmetry to perform a suppression subtractive hybridization screening to select for transcripts preferentially expressed in the developing left ovary as compared to the right one, and thus identify genes that are potentially involved in the process of ovarian differentiation. Results: One of these transcripts, named Ovex1 according to its expression profile, corresponds to an endogenous retrovirus that has not been previously characterized. It is transcribed as full-length and singly spliced mRNAs and contains three uninterrupted open reading frames coding potentially for proteins with homology to Gag and Pro-Pol retroviral polyproteins and a third protein showing only a weak similarity with Env glycoproteins. Ovex1 is severely degenerated; it is devoid of typical long terminal repeats and displays some evidence of recombination. An orthologous Ovex1 locus was identified in the genome of zebra finch, a member of a different bird order, and similar sequences were detected in turkey, guinea fowl, and duck DNA. The relationship between these sequences follows the bird phylogeny, suggesting vertical transmission of the endogenous retrovirus for more than 100 million years.Ovex1 is transcribed in chicken gonads with a sex-dependent and left-right asymmetrical pattern. It is first expressed in the cortex of the left indifferent gonads of both sexes. Expression is transient in the left testis and absent in the right one. In developing ovaries, Ovex1 transcription increases sharply in the left cortex and is weakly detected in the medulla. After folliculogenesis, Ovex1-expressing cells constitute the follicular granulosa cell layer. Ovex1 expression highlights a striking desquamation process that leads to profound cortical remodeling associated with follicle morphogenesis. Conclusions: Evidence for a selection pressure at the protein level suggests that this endogenous retrovirus, expressed in the ovarian supporting cell lineage, might play an active role in bird ovarian physiology.

Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina), a primate species susceptible to Human Immunodeficiency Virus type 1 infection

Yi-Qun Kuang — 2009-06-09T00:00:00Z

Background: The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We have previously reported that the TRIM5-Cyclophilin A (TRIMCyp) fusion in pig-tailed macaques (Macaca nemestrina) is dysfunctional in restricting HIV-1, which may explain why pig-tailed macaques are susceptible to HIV-1 infection. Similar results have also been reported by other groups. However, according to the current primate taxonomy, the previously reported M. nemestrina are further classified into three species, which all belong to the Macaca spp. This calls for the need to look into the previous studies in more details. Results: The local species Northern pig-tailed macaque (M. leonina) was analyzed for the correlation of TRIM5 structure and HIV-1 infection. Eleven M. leonina animals were analyzed, and all of them were found to possess TRIM5-CypA fusion at the TRIM5 locus. The transcripts encoding the dysfunctional TRIM5-CypA should result from the G-to-T mutation in the 3'-splicing site of intron 6. Polymorphism in the putative TRIMCyp recognition domain was observed. The peripheral blood mononuclear cells (PBMCs) of M. leonina were susceptible to HIV-1 infection. Consistent with the previous results, expression of the M. leonina TRIMCyp in HeLa-T4 cells rendered the cells resistant to HIV-2ROD but not to SIVmac239 infection. Conclusion: The susceptibility of M. leonina to HIV-1 infection is due to the dysfunctional TRIM5-CypA fusion in the TRIM5 locus. This finding should broaden our perspective in developing better HIV/AIDS non-human primate animal models.

Nef gene evolution from a single transmitted strain in acute SIV infection

Benjanmin Bimber — 2009-06-08T00:00:00Z

Background: The acute phase of immunodeficiency virus infection plays a crucial role in determining steady-state virus load and subsequent progression of disease in both humans and nonhuman primates. The acute period is also the time when vaccine-mediated effects on host immunity are likely to exert their major effects on virus infection. Recently we developed a Monte-Carlo (MC) simulation with mathematical analysis of viral evolution during primary HIV-1 infection that enables classification of new HIV-1 infections originating from multiple versus single transmitted viral strains and the estimation of time elapsed following infection. Results: A total of 322 SIV nef SIV sequences, collected during the first 3 weeks following experimental infection of two rhesus macaques with the SIVmac239 clone, were analyzed and found to display a comparable level of genetic diversity, 0.015% to 0.052%, with that of env sequences from acute HIV-1 infection, 0.005% to 0.127%. We confirmed that the acute HIV-1 infection model correctly identified the experimental SIV infections in rhesus macaques as "homogenous" infections, initiated by a single founder strain. The consensus sequence of the sampled strains corresponded to the transmitted sequence as the model predicted. However, measured sequential decrease in diversity at day 7, 11, and 18 post infection violated the model assumption, neutral evolution without any selection. Conclusion: While nef gene evolution over the first 3 weeks of SIV infection originating from a single transmitted strain showed a comparable rate of sequence evolution to that observed during acute HIV-1 infection, a purifying selection for the founder nef gene was observed during the early phase of experimental infection of a nonhuman primate.

Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

Yeshitila Friew — 2009-06-04T00:00:00Z

Background: Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments. Results: The results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion: These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.

96 shRNAs designed for maximal coverage of HIV-1 variants

Glen John Mcintyre — 2009-06-04T00:00:00Z

Background: The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains. Results: We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 – 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains. Conclusion: In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

Multiple-infection and recombination in HIV-1 within a longitudinal cohort of women

Alan Templeton — 2009-06-03T00:00:00Z

Background: Recombination between strains of HIV-1 only occurs in individuals with multiple infections, and the incidence of recombinant forms implies that multiple infection is common. Most direct studies indicate that multiple infection is rare. We determined the rate of multiple infection in a longitudinal study of 58 HIV-1 positive participants from The Women's Interagency HIV Study with a richer sampling design than previous direct studies, and we investigated the role of recombination and sampling design on estimating the multiple infection rate. Results: 40% of our sample had multiple HIV-1 infections. This rate of multiple infection is statistically consistent with previous studies once differences in sampling design are taken into account. Injection drug use significantly increased the incidence of multiple infections. In general there was rapid elimination of secondary strains to undetectable levels, but in 3 cases a superinfecting strain displaced the initial infecting strain and in two cases the strains coexisted throughout the study. All but one secondary strain was detected as an inter- and/or intra-genic recombinant. Injection drug use significantly increased the rate of observed recombinants. Conclusion: Our multiple infection rate is consistent with rates estimated from the frequency of recombinant forms of HIV-1. The fact that our results are also consistent with previous direct studies that had reported a much lower rate illustrates the critical role of sampling design in estimating this rate. Multiple infection and recombination significantly add to the genetic diversity of HIV-1 and its evolutionary potential, and injection drug use significantly increases both.

Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species

Kei Sato — 2009-06-02T00:00:00Z

In this study, we first demonstrate that endogenous hBST-2 is predominantly expressed on the plasma membrane of a human T cell line, MT-4 cells, and that Vpu-deficient HIV-1 was less efficiently released than wild-type HIV-1 from MT-4 cells. In addition, surface hBST-2 was rapidly down-regulated in wild-type but not Vpu-deficient HIV-1-infected cells. This is a direct insight showing that provirus-encoded Vpu has the potential to down-regulate endogenous hBST-2 from the surface of HIV-1-infected T cells. Corresponding to previous reports, the aforementioned findings suggested that hBST-2 has the potential to suppress the release of Vpu-deficient HIV-1. However, the molecular mechanism(s) for tethering HIV-1 particles by hBST-2 remains unclear, and we speculated about the requirement for cellular co-factor(s) to trigger or assist its tethering ability. To explore this possibility, we utilize several cell lines derived from various species including human, AGM, dog, cat, rabbit, pig, mink, potoroo, and quail. We found that ectopic hBST-2 was efficiently expressed on the surface of all analyzed cells, and its expression suppressed the release of viral particles in a dose-dependent manner. These findings suggest that hBST-2 can tether HIV-1 particles without the need of additional co-factor(s) that may be expressed exclusively in primates, and thus, hBST-2 can also exert its function in many cells derived from a broad range of species. Interestingly, the suppressive effect of hBST-2 on HIV-1 release in Vero cells was much less pronounced than in the other examined cells despite the augmented surface expression of ectopic hBST-2 on Vero cells. Taken together, our findings suggest the existence of certain cell types in which hBST-2 cannot efficiently exert its inhibitory effect on virus release. The cell type-specific effect of hBST-2 may be critical to elucidate the mechanism of BST-2-dependent suppression of virus release.

"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

Andrea Savarino — 2009-06-02T00:00:00Z

Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the eradication of HIV-1. For this purpose, "shock and kill" strategies have been proposed (activation of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs) induce HIV-1 activation from quiescence, yet class/isoform-selective HDACIs are needed to specifically target HIV-1 latency. We tested 32 small molecule HDACIs for their ability to induce HIV-1 activation in the ACH-2 and U1 cell line models. In general, potent activators of HIV-1 replication were found among non-class selective and class I-selective HDACIs. However, class I selectivity did not reduce the toxicity of most of the molecules for uninfected cells, which is a major concern for possible HDACI-based therapies. To overcome this problem, complementary strategies using lower HDACI concentrations have been explored. We added to class I HDACIs the glutathione-synthesis inhibitor buthionine sulfoximine (BSO), in an attempt to create an intracellular environment that would facilitate HIV-1 activation. The basis for this strategy was that HIV-1 replication decreases the intracellular levels of reduced glutathione, creating a pro-oxidant environment which in turn stimulates HIV-1 transcription. We found that BSO increased the ability of class I HDACIs to activate HIV-1. This interaction allowed the use of both types of drugs at concentrations that were non-toxic for uninfected cells, whereas the infected cell cultures succumbed more readily to the drug combination. These effects were associated with BSO-induced recruitment of HDACI-insensitive cells into the responding cell population, as shown in Jurkat cell models for HIV-1 quiescence. The results of the present study may contribute to the future design of class I HDACIs for treating HIV-1. Moreover, the combined effects of class I-selective HDACIs and the glutathione synthesis inhibitor BSO suggest the existence of an Achilles' heel that could be manipulated in order to facilitate the "kill" phase of experimental HIV-1 eradication strategies.