Accuracy estimation of foamy virus genome copying

<p>Abstract</p> <p>Background</p> <p>Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the <it>in vitro </it>error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying <it>in vivo </it>we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.</p> <p>Results</p> <p>We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an <it>in vivo </it>error rate of approximately 4 × 10^-4per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral <it>bet </it>gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate <it>in vivo</it>. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic <it>in vivo </it>error rate of 1.1 × 10^-5per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.</p> <p>Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.</p> <p>Conclusion</p> <p>Our results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.</p

AZT-resistant foamy virus

AbstractAzidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness

The Role of Caveolin 1 in HIV Infection and Pathogenesis

Caveolin 1 (Cav-1) is a major component of the caveolae structure and is expressed in a variety of cell types including macrophages, which are susceptible to human immunodeficiency virus (HIV) infection. Caveolae structures are present in abundance in mechanically stressed cells such as endothelial cells and adipocytes. HIV infection induces dysfunction of these cells and promotes pathogenesis. Cav-1 and the caveolae structure are believed to be involved in multiple cellular processes that include signal transduction, lipid regulation, endocytosis, transcytosis, and mechanoprotection. Such a broad biological role of Cav-1/caveolae is bound to have functional cross relationships with several molecular pathways including HIV replication and viral-induced pathogenesis. The current review covers the relationship of Cav-1 and HIV in respect to viral replication, persistence, and the potential role in pathogenesis

Black African immigrants in Australia : an exploratory analysis of the impacts of race and class on their lived experiences and adaptation processes

Deposited with permission of the author. © 2005 Dr. Ayalew MergiaThe primary aim of the present study is to explore the impact of race and class on the lived experiences of Black Africans in Melbourne during their adaptation process as Black immigrants in the historical, socio-cultural, economic, and political context of Australia. Critical ethnography is the methodology for this study. Focussing directly on the everyday lives of Black African immigrants over a twenty-three months period during February 2003 to end of January 2005, the study reveals how racial factors articulate and intersect with class factors in the making and/or shaping of the lived experiences of Black Africans in Melbourne, Australia. The study uses a definition of race that focuses on the demarcation of `races' as socio-historical constructions embedded in power relations that enable the majority to define the identities of racially defined minorities, definitions that serve to reinforce and perpetuate the minority group's inferior social status. The mean age of the participants was 36.9 years (SD = 8.6). The participants were diverse in their manner of immigration, motivations for immigration, and household characteristics. Interviews, participant-observation, and paper-and-pencil tools (survey questionnaires) were the three methods used for data collection. Riesman's narrative analysis (1993) is the framework for analysing the interview data. Quantitative data provided descriptive information on demographic and immigration profiles, socioeconomic and housing characteristics. The orientation of the quantitative component was to obtain a range of pictures of information or knowledge distributed within the population, and not to attempt any generalisation from the participants to the entire population of such immigrants. Findings from the traditional analysis indicated that participants faced immediate issues of survival after they arrived in Melbourne. Findings from the critical analysis revealed that immigration policy, class, imperialism and capitalism are not abstract or concealed issues but rather very real ones that have a direct bearing on these immigrants' experiences and their capacities to adapt. Findings from the critical analysis reveal that Black African immigrants received mistreatment from both white Australians (the dominant group) and non-English speaking Europeans (formerly oppressed groups). This study extends our understanding of immigrants' lived experiences; at the same time, it raises more questions for further investigation. In addition to the substantive contribution, this study also draws attention to several methodological issues pertinent to bilingual immigrant studies

Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I

Abstract Background HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells. Results Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased. Conclusions These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV’s persistent infection of macrophages.</p