Evolutionary variation of papillomavirus E2 protein and E2 binding sites

Background: In an effort to identify the evolutionary changes relevant to E2 function, within and between papillomavirus genera, we evaluated the E2 binding sites (E2BS)s inside the long-control-region (LCR), and throughout the genomes. We identified E2BSs in the six largest genera of papillomaviruses: Alpha, Beta, Gamma, Delta, Lambda, and Xi-papillomaviruses (128 genomes), by comparing the sequences with a model consensus we created from known functional E2BSs (HPV16, HPV18, BPV1). We analyzed the sequence conservation and nucleotide content of the 4-nucleotide spacer within E2BSs. We determined that there is a statistically significant difference in GC content of the four-nucleotide E2BS spacer, between Alpha and Delta-papillomaviruses, as compared to each of the other groups. Additionally, we performed multiple alignments of E2 protein sequences using members of each genus in order to identify evolutionary changes within the E2 protein. Results: When a phylogenetic tree was generated from E2 amino acid sequences, it was discovered that the alpha-papillomavirus genera segregates into two distinct subgroups (α1 and α2). When these subgroups were individually analyzed, it was determined that the subgroup α1 consensus E2BS favored a spacer of AAAA, whereas subgroup α2 favored the opposite orientation of the same spacer; TTTT. This observation suggests that these conserved inverted linkers could have functional importance

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background: The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. Results: We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. Conclusions: These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication

Characterization of Binding and Fusion Efficiencies Mediated by the V1-V5 Env Derived from Transmitted and Non-transmitted Viruses Isolated from a Perinatal Transmission Cohort from Zambia

Human Immunodeficiency Virus Type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS), which affects over 34 million people worldwide. In sub-Saharan Africa where access to antiretroviral therapies (ART) is limited, mother-to-child transmission (MTCT) rates remain high and represent a major concern in the global HIV/AIDS epidemic. Little is known about the biological properties of viruses that are transmitted perinatally, including how the biological functions of envelope (Env) influence transmissibility. Previously, transmitted viruses were found to have an advantage in replicative fitness mediated by Env V1-V5. In this study viruses derived from transmitting mother infant pairs (MIPs) were used to determine if the binding and fusion activities are influenced by Env V1-V5 and if any differences correlate to replicative fitness and transmission. Fusion and binding assays were used to measure the biological functions of Env from individual clones of five MIP. RESULTS: Neither binding nor fusion is predictive of transmission. However, clonal variation in both functions is observed, demonstrating V1-V5 is capable of influencing fusion and binding. Fusion correlates with infectivity, but does not correlate with binding. The results of this study have provided new insights to better understand the functional properties of Env and its role in MTCT. Advisor: Charles Woo

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Abstract Background The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. Results We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. Conclusions These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication.</p

Comparative analysis of the fusion efficiency elicited by the envelope glycoprotein V1–V5 regions derived from human immunodeficiency virus type 1 transmitted perinatally

Understanding the properties of viruses preferentially establishing infection during perinatal transmission of human immunodeficiency virus type 1 (HIV-1) is critical for the development of effective measures to prevent transmission. A previous study demonstrated that the newly transmitted viruses (in infants) of chronically infected mother–infant pairs (MIPs) were fitter in terms of growth, which was imparted by their envelope (Env) glycoprotein V1–V5 regions, than those in the corresponding chronically infected mothers. In order to investigate whether the higher fitness of transmitted viruses was conferred by their higher entry efficiency directed by the V1–V5 regions during perinatal transmission, the fusogenicity of Env containing V1–V5 regions derived from transmitted and nontranmsmitted viruses of five chronically infected MIPs and two acutely infected MIPs was analysed using two different cell–cell fusion assays. The results showed that, in one chronically infected MIP, a higher fusion efficiency was induced by the infant Env V1–V5 compared with that of the corresponding mother. Moreover, the V4–V5 regions played an important role in discriminating the transmitted and non-transmitted viruses in this pair. However, neither a consistent pattern nor significant differences in fusogenicity mediated by the V1–V5 regions between maternal and infant variants was observed in the other MIPs. This study suggests that there is no consistent and significant correlation between viral fitness selection and entry efficiency directed by the V1–V5 regions during perinatal transmission. Other factors such as the route and timing of transmission may also be involved