Identification and investigation of cellular factors associated with HIV replication

Human immunodeficiency virus type-1 (HIV-1) is a member of the lentivirus subfamily of the retroviridae family. Currently, HIV infection is considered incurable. Combinations of antiretroviral therapies will limit HIV replication and is able to extend the patients' lives. However, HIV-1 multi-drug resistance continues to rise. Therefore, novel HIV inhibitors are needed. Like all viruses, HIV-1 requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. The first study of this dissertation is based on a previous proteomic screening in our laboratory that identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in HIV-1 replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1, which is required for Tat-dependent transactivation of viral gene expression. In this study, the roles of NF45 and NF90 in HIV replication were investigated through over-expression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA in a sequence independent manner. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies demonstrate that both NF45 and NF90 potentiate HIV infection through their RNA binding activities. In the next study described in this dissertation, I constructed a labelled virus to perform affinity purification-mass spectrometry and identify the host factors which interact with the matrix (MA) protein of HIV. A Strep affinity tag was inserted into the C-terminus of the MA protein. The resultant virus was replication competent and used to infect Jurkat T-cells. Matrix complexes were affinity purified with Strep-Tactin agarose. Protein identification was performed by sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry. The data was log2 transformed and student t-tests with Bonferroni correction used to determine proteins associated with matrix. A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. Ku70, Ku80 and YB-1 were confirmed to interact with matrix by immunoblot. Knockdown of two candidates, EZRIN and YB-1, enhanced HIV infection in vitro. These data further increased our understanding of HIV-host cell interactions. For the final study, we investigated the effects of deletions in the C-terminal alpha helix (?5) of MA on virus assembly release. Deletion of the N-terminus of ?5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA?96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA?96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the ?5 of MA inhibits virus release.