Project description:To investigate how the major HIV-1 structural protein Gag packages viral genomes efficiently, we replaced the NC domain of Gag with RNA binding domains from heterologous cellular RNA binding proteins to generate Gag chimeras. We hypothesize that the Gag chimeras that preferentially bind to purine-rich RNA sequences will package viral genomes efficiently. We performed CLIP-seq to identify the RNA sequences bound by WT Gag and Gag chimeras in cells, at the plasma membrane, and in virions.
Project description:To investigate how the major HIV-1 structural protein Gag packages viral genomes efficiently, we replaced the NC domain of Gag with RNA binding domains from heterologous cellular RNA binding proteins to generate Gag chimeras. We hypothesize that the Gag chimeras that preferentially bind to purine-rich RNA sequences will package viral genomes efficiently. We performed CLIP-seq to identify the RNA sequences bound by WT Gag and Gag chimeras in cells, at the plasma membrane, and in virions.
Project description:To investigate how the major HIV-1 structural protein Gag packages viral genomes efficiently, we replaced the NC domain of Gag with RNA binding domains from heterologous cellular RNA binding proteins to generate Gag chimeras. We hypothesize that the Gag chimeras that preferentially bind to purine-rich RNA sequences will package viral genomes efficiently. We performed CLIP-seq to identify the RNA sequences bound by WT Gag and Gag chimeras in cells, at the plasma membrane, and in virions.
Project description:The HIV-1 Gag protein orchestrates all steps of virion genesis, including RNA recruitment into virions. However, the identities of specific RNA sequences recognized by Gag in cells and virions are largely unknown. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover dramatic changes in the RNA binding specificity of Gag during virion genesis that are induced by its membrane binding, multimerization and proteolytic maturation. Prior to assembly, and also in mature virions, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during assembly of immature virions, this specificity changes in a manner that facilitates genome packaging, as nucleocapsid binds to many sites on the viral genome and mRNA sequences with a similar A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.
Project description:We performed a 3' RACE of a novel HIV RNA TAR-gag in order to determine the sequence of the RNA at the 3' end. Our data had shown that TAR-gag was potentially a noncoding RNA and our hypothesis was that TAR-gag ended somewhere prior to the end of the gag region of the HIV genome. The 3' RACE experiment showed that TAR-gag actually consists of four different RNA clusters, the longest of which ends at 615 bases from the transcription start site; this is in the middle of the p17 region of the gag gene. In addition, we sequenced all host RNAs in the EVs.
Project description:We analyzed the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions and observed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only five miRNAs were found at a 2 to 4-fold higher level in virions than predicted based on random sampling. Of note, these included two miRNAs, miR-155 and miR-92a, reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding induces virion incorporation, we introduced artificial miRNA target sites into the HIV-1 genome and observed a 10 to 40-fold increase in the packaging of the cognate miRNA into virions, leading to the recruitment of up to 1.6 copies into each virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. We conclude that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to restrict cellular miRNA binding to their genome.
Project description:Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes. This dataset contains results from a mass spectrometry approach using affinity-tagged (His6) HIV-1 and RSV Gag proteins mixed with nuclear extracts, and identifies potential binding partners of HIV-1 and RSV Gag involved in several nuclear processes, including transcription, splicing, RNA modification, and chromatin structure. Although a subset of host proteins interacted with both Gag proteins, there were also unique host proteins belonging to each interactome dataset. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.
Project description:Integrase CLIP-seq experiments were conducted on wild-type and eccentric HIV-1 virions generated in the presence of allosteric integrase inhibitors and IN K264/266A and R269/K273A mutations