By the end of this year, the World Health Organization has estimated that approximately 0.7% of the world's population will be seropositive for human immunodeficiency virus (HIV). Most of these individuals live in the developing world and do not have access to highly active anti-retroviral therapy (HAART). A safe and efficacious vaccine may be a decade away. Most therapy is directed towards inhibition of viral reverse transcriptase or protease. Although over the last two decades much has been learned regarding the replicative cycle of HIV and the cellular factors involved, there are still gaps in our knowledge that could represent future therapeutic targets. For example, in the mouse entry and post-entry blocks to HIV replication have been circumvented by expressing human CD4, a chemokine co-receptor, and cyclin T1. These mouse cells, however, are still not fully permissive for HIV replication, perhaps due to a defect in Gag processing. Mouse-human cell fusions produce infectious virus, suggesting that mouse cells lack one or more factors required for HIV replication. This proposal seeks by genetic means to identify novel host cellular factors that may facilitate HIV release, using poorly permissive rodent cells as a model. In the first aim, a genetic screen will be used to identify such a missing factor(s). A cDNA library expression vector has been developed based upon HIV which is of high titer and has a selectable marker. This will be introduced into mouse cells expressing cyclin T1 (cycT1). Infectious virus will be recovered by providing trans functions, titered, and used to re-transduce na'fve mouse.cycT1 cells. This cycle will then be repeated in the hopes of amplifying and enriching for cDNAs that facilitate HIV replication in the mouse. In the second aim, a conventional plasmid-based cDNA library will be introduced into mouse cells (already expressing cycT1 and transduced with an HIV vector encoding both luciferase and a selectable marker). Cells will be divided into pools, and infectious virus recovered from each pool by methods similar to aim 1. Cell clones supporting the highest degree of HIV release will be isolated by sib selection. For both this and the preceding aim, cDNAs from individual clones will be recovered by PCR, characterized by DNA sequencing, and re-introduced into mouse cells to determine if the mouse cells are then rendered fully permissive for HIV replication. In the third aim, the Stanford panel of radiation hybrid (RH) cell lines will be individually transduced with a replication-defective HIV vector encoding cycT1 and a marker. Infectious virus will be recovered from each RH by methods similar to aim 1. The most promising RHs will be tested for their ability to rescue HIV Gag processing and support wild-type M-tropic HIV replication similar to the first two aims. This RH panel should allow functional genetic mapping to less than 1 Mb. From this aim it is hoped that a reasonable approximation of the number (and chromosomal location) of missing host factors will be obtained and this will be correlated with the results from the first two aims. Time permitting, candidate genes from the region of greatest interest will be tested for their ability to mediate HIV release from mouse cells. At the completion of these studies it is hoped that a better understanding of the host factors involved in HIV release will be achieved.
| Coskun, Ayse K; van Maanen, Marc; Janka, David et al. (2007) Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release. Virology 362:283-93 |
