APOBEC3 proteins, in particular APOBEC3G/F, are important innate host factors that contribute to protection from HIV-1 infection by inducing high levels of guanine to adenine nucleotide substitutions (termed hypermutation) during HIV-1 viral replication. These nucleotide substitutions occur at different rates and locations across the HIV-1 genome and are thought to be particularly more frequent in the pol region. The virus has evolved ways to counteract these host factors by inducing degradation of APOBEC3G/F proteins through protein interactions with HIV-1 Vif. The aim of this thesis is to characterize and investigate the role of APOBEC3G/F-mediated hypermutation in the HIV-1 genome. We identified a subset of women from the Pumwani Commercial Sex Worker (CSW) cohort with significantly higher rates of hypermutated proviruses in pol. This degree of hypermutation correlated to less severe HIV disease progression as measured by CD4+ T cell count. This was in agreement with previous studies that evidence of APOBEC-mediated hypermutation correlate with reduced disease progression, confirming APOBEC3G/F proteins role in HIV-1 disease. Furthermore, we investigated the in vitro and ex vivo interaction between HIV-1 Vif and APOBEC3G from subjects infected with hypermutated and non-hypermutated proviruses. In vitro studies indicated that HIV-1 Vif protein expression in subjects with hypermutated proviruses were quite divergent and levels of APOBEC3G also differed between subjects. Ex vivo studies in subjects with hypermutated proviruses indicated that endogenous APOBEC3G expression was greater than in subjects with hypermutated proviruses. Both studies suggest that host and viral factors such as APOBEC3G and HIV-1 Vif are playing an influential role in HIV-1 pathogenesis. Further investigations into these interactions may lead to novel strategies into the development of therapeutic drugs for the fight against HIV-1.

HIV-1 integrase (IN) enzyme employs several viral and cellular proteins for nuclear translocation and crucial integration of viral cDNA. Successful identification of new viral/cellular interactions may shed light for better understanding of HIV-1 replication. 293T cells were transiently transfected with pYEF-1-TAP-IN and cell lysate were subjected to Tandem Affinity Purification system to pull down putative IN-interacting cellular partners. A number of distinct bands from the Coomassie-stained gel were excised followed by in-gel digestion and mass spectrometry. Putative cellular partners of HIV-1 IN were heat shock protein 60 (HSP60), β-tubulin, γ-actin, ATP synthase alpha subunit and histone H1.2 were identified by mass spectrometry. Additionally, SF3A3 (splicing factor 3A3), another previously reported factor, was successfully co-immunoprecipitated with IN. The C-terminal portion of IN was found to be the region of interaction with SF3A3. Overall, this study has provided better understanding of IN dynamics enriching existing knowledge of HIV-1 IN biology.

Objective: Serpins are serine protease inhibitors that are involved in a wide variety of biological functions in nature. They are known to regulate inflammation processes as well as provide host defense against microorganisms. Recent evidence has associated many types of mucosal serpins with a protective phenotype against HIV infection in women. Our hypothesis is that serpins with known antiviral activity against HIV-1 are correlated with protection in a group of HIV exposed seronegative individuals (HIV-resistant) from the Pumwani sex worker cohort. Study design: Cervico-vaginal lavage (CVL) fluid was collected from 66 HIV-positive, 82 HIV-negative and 84 HIV-resistant sex workers from the cohort. Clinical and epidemiological information was recorded at the time of sample collection. CVL protein levels were determined by BCA assay and serpin (A1 and A3) concentrations by a commercially available ELISA kit. Mucosal serpin concentrations were compared against clinical and epidemiological factors as well as sexual practices. Results: Serpin A1 was significantly higher in the HIV-resistant group compared to the HIV-negative controls (Anova: p=0.0470*). Total concentration of serpin A3 did not reach statistical significance between groups. Serpins did not correlate with age, sexual practices, contraceptive use or number of pregnancies. Serpins were differentially abundant during different stages of the menstrual cycle whereas serpin A1 was elevated during the proliferation phase but not in secretory phase (p=0.0275*). Conclusion: Serpin A1 was correlated with HIV-protection in this group of HESN women. This work will contribute to a more complete understanding of mechanisms of resistance and susceptibility to HIV infection.

HIV-1 integrase (IN) enzyme employs several viral and cellular proteins for nuclear translocation and crucial integration of viral cDNA. Successful identification of new viral/cellular interactions may shed light for better understanding of HIV-1 replication. 293T cells were transiently transfected with pYEF-1-TAP-IN and cell lysate were subjected to Tandem Affinity Purification system to pull down putative IN-interacting cellular partners. A number of distinct bands from the Coomassie-stained gel were excised followed by in-gel digestion and mass spectrometry. Putative cellular partners of HIV-1 IN were heat shock protein 60 (HSP60), β-tubulin, γ-actin, ATP synthase alpha subunit and histone H1.2 were identified by mass spectrometry. Additionally, SF3A3 (splicing factor 3A3), another previously reported factor, was successfully co-immunoprecipitated with IN. The C-terminal portion of IN was found to be the region of interaction with SF3A3. Overall, this study has provided better understanding of IN dynamics enriching existing knowledge of HIV-1 IN biology.

APOBEC3 proteins, in particular APOBEC3G/F, are important innate host factors that contribute to protection from HIV-1 infection by inducing high levels of guanine to adenine nucleotide substitutions (termed hypermutation) during HIV-1 viral replication. These nucleotide substitutions occur at different rates and locations across the HIV-1 genome and are thought to be particularly more frequent in the pol region. The virus has evolved ways to counteract these host factors by inducing degradation of APOBEC3G/F proteins through protein interactions with HIV-1 Vif. The aim of this thesis is to characterize and investigate the role of APOBEC3G/F-mediated hypermutation in the HIV-1 genome. We identified a subset of women from the Pumwani Commercial Sex Worker (CSW) cohort with significantly higher rates of hypermutated proviruses in pol. This degree of hypermutation correlated to less severe HIV disease progression as measured by CD4+ T cell count. This was in agreement with previous studies that evidence of APOBEC-mediated hypermutation correlate with reduced disease progression, confirming APOBEC3G/F proteins role in HIV-1 disease. Furthermore, we investigated the in vitro and ex vivo interaction between HIV-1 Vif and APOBEC3G from subjects infected with hypermutated and non-hypermutated proviruses. In vitro studies indicated that HIV-1 Vif protein expression in subjects with hypermutated proviruses were quite divergent and levels of APOBEC3G also differed between subjects. Ex vivo studies in subjects with hypermutated proviruses indicated that endogenous APOBEC3G expression was greater than in subjects with hypermutated proviruses. Both studies suggest that host and viral factors such as APOBEC3G and HIV-1 Vif are playing an influential role in HIV-1 pathogenesis. Further investigations into these interactions may lead to novel strategies into the development of therapeutic drugs for the fight against HIV-1.

Objective: Serpins are serine protease inhibitors that are involved in a wide variety of biological functions in nature. They are known to regulate inflammation processes as well as provide host defense against microorganisms. Recent evidence has associated many types of mucosal serpins with a protective phenotype against HIV infection in women. Our hypothesis is that serpins with known antiviral activity against HIV-1 are correlated with protection in a group of HIV exposed seronegative individuals (HIV-resistant) from the Pumwani sex worker cohort. Study design: Cervico-vaginal lavage (CVL) fluid was collected from 66 HIV-positive, 82 HIV-negative and 84 HIV-resistant sex workers from the cohort. Clinical and epidemiological information was recorded at the time of sample collection. CVL protein levels were determined by BCA assay and serpin (A1 and A3) concentrations by a commercially available ELISA kit. Mucosal serpin concentrations were compared against clinical and epidemiological factors as well as sexual practices. Results: Serpin A1 was significantly higher in the HIV-resistant group compared to the HIV-negative controls (Anova: p=0.0470*). Total concentration of serpin A3 did not reach statistical significance between groups. Serpins did not correlate with age, sexual practices, contraceptive use or number of pregnancies. Serpins were differentially abundant during different stages of the menstrual cycle whereas serpin A1 was elevated during the proliferation phase but not in secretory phase (p=0.0275*). Conclusion: Serpin A1 was correlated with HIV-protection in this group of HESN women. This work will contribute to a more complete understanding of mechanisms of resistance and susceptibility to HIV infection.

This thesis project was designed to investigate the phenotypic and genotypic variability of human immunodeficiency virus type 1 (HIV-1) drug-naive clade C reverse transcriptase (RT) and its potential impact in the development of resistance against inhibitors of RT. Five treatment-naive HIV-1 Ethiopian isolates were classified as subtype C on the basis of env gene heteroduplex mobility assays (HMA) profile and phylogenetic analysis of RT sequences. In subtype C RT, a specific KVEQ motif of silent mutations (amino acid 65, 106, 138, 161) at resistance sites was present. Two Ethiopian strains were naturally resistant to non-nucleoside RT inhibitors (NNRTI), as well as to zidovudine (ZDV), based on the natural polymorphisms of G190A and K70R, respectively. The final drug concentration that selected for NNRTI primary resistance mutations in tissue culture assays was significantly higher for clade B than clade C for each of nevirapine (NW) (10 muM versus 2 or 4 muM), efavirenz (EFV) (1muM versus 0.01muM) and delaviridine (DLV) (10muM versus 1 or 4muM), respectively. In the middle of the selection period with all the NNRTIs, subtype B viruses were harboring a mixture of both wild type and mutated forms, whereas in clade C viruses, primary resistance mutations were fully generated. Thus, we have found that clade C isolates developed more rapidly resistance (8 or 9 weeks with NVP or DLV and 13 weeks with EFV) as compared with clade B controls (at least 15 weeks with NW or DLV and 30 weeks with EFV). Odd mutations were detected during selection with NNRTIs, such as S98I, and two mutations (A62V and V75E), at sites associated to multi-drug resistance against nucleoside inhibitors (NRTIs). The substitution A62V was initially observed as a drug-naive silent mutation A62A. NW and DLV mutants were broadly cross-resistants. Following in vitro selection for drug-resistance with NNRTIs (NVP, DLV and EFV) and NRTIs [lamivudine (3TC) and ZDV], RT immunodominant regions of 14 HIV-1 s

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

A infecção pelo vírus da imunodeficiência humana (HIV) foi largamente disseminada pela população humana a partir da década de 80. Diversos estudos retrocedem o surgimento da epidemia do HIV a vírus infectantes de símios africanos de vida livre e ao conseqüente contato entre estas espécies e humanos. As rápidas taxas evolutivas e a associação às inter-relações humanas foram suficientes para a disseminação e a diversificação das formas virais. Desta forma, a expansão do HIV-1 pelo mundo estabeleceu uma pandemia formada por diferentes variantes virais. O subtipo B foi a primeira forma detectada do vírus e atualmente está presente em um grande número de países, sendo responsável por aproximadamente 10% das infecções por HIV no mundo todo. Fora da África, o subtipo B emergiu na região do Caribe, se disseminou pelos países vizinhos e atingiu os Estados Unidos, onde estabeleceu a pandemia. O traçado geográfico das rotas de disseminação nas Américas a partir do Haiti é, até o momento, difuso ou ausente. O presente trabalho visa investigar a história evolutiva do subtipo B nas Américas e, através de filogenias e análises estatísticas, examinar o papel da América do Sul no estabelecimento da epidemia. Os resultados obtidos sugerem uma participação primária das populações da América do Sul na disseminação do subtipo B. A estruturação populacional e as filogenias corroboram uma ligação epidemiológica importante entre os países do Caribe e os da América do Sul, visto que um clado agrupando isolados sul-americanos emerge diretamente do clado caribenho. Além deste, um grande grupo, formado por sequências das três Américas, denominado cluster pandêmico, compartilha um ancestral comum com o clado da América do Sul. Possíveis explicações para estes resultados envolvem a co-dispersão para as Américas a partir do Caribe e também a intermediação da América do Sul entre o Caribe e os Estados Unidos no estabelecimento da pandemia. Dados históricos de migrações populacionais a partir do Caribe reforçam estes cenários. Os dados apresentados revelam uma nova perspectiva a respeito da epidemia do HIV-1 subtipo B. Além disso, destacam a utilidade de uma abordagem populacional para o entendimento da dispersão da epidemia, contribuindo para a avaliação entre a diversidade viral e a movimentação de populações humanas. / Infections with human immunodeficiency virus (HIV) have been widely disseminated by the human population from the 1980's. Several studies point the onset of the epidemic to virus infecting free-living African apes through blood contact between these species and humans. The fast evolutionary rates and the human relationships were sufficient for the spreading and diversification of the epidemic. Thus, the spread oh HIV-1 established a worldwide pandemic formed by different viral forms. Among them, subtype B was the first form detected and is currently present in a large number of countries, accounting for approximately 10% of HIV infections worldwide. Out of Africa, subtype B likely emerged in Haiti in the Caribbean region, reached neighboring countries and the United States, where the pandemic was established. The geographical layout of the routes of spread within the Americas, so far, is diffuse or absent. This study aimed to investigate the evolutionary history of subtype B in the Americas and, through phylogenetic and statistical analyses sought to examine the role of South America in the pandemic. Our main findings suggest a primary involvement of the populations of South America in the spread of subtype B. The genetic structure of the viral population and the phylogenetic analyses supported an epidemiological link among the countries of the Caribbean and South American ones since a clade grouping isolates from South America emerges directly from the Caribbean. Besides this, a large group, formed by sequences of the three Americas, called pandemic cluster, share a common ancestor with the clade of South America. The co-dispersion to the Americas from the Caribbean and the intermediation of South America between the Caribbean and North America epidemics are explanations to support our results. The data presented here outline a new perspective on the HIV-1 subtype B epidemic. Furthermore, we highlight the usefulness of a population-based approach to understanding the spread of the epidemic, contributing to the assessment between the viral diversity and the movement of human populations.