Retroviral Reverse Transcription
My laboratory studies preintegrative steps in retroviral replication using Moloney murine leukemia virus (M-MuLV) as a model system. We are particularly interested in features of reverse transcriptase and the viral genomic RNA that determine the form and functionality of retroviral DNA.
One area of research involves analyzing the pathway of retroviral DNA synthesis within infected cells. Interestingly, although it is known that very few of the retroviral particles that attach to cells ultimately generate integrated DNAs, the efficiencies of the preintegrative steps in retroviral replication have not been well-studied. We are developing assays to quantify retroviral integration and the reverse transcription steps that precede it in order to examine how genetic and environmental variation affects the early stages of retroviral replication.
Another key research direction is aimed at understanding how structural and organizational properties of retroviral genomes affect the outcome of retroviral replication. Although the usual size and organization of retroviral genomes is well known, the driving forces that determine this form and limitations to deviation from it are unknown and are critical in understanding the potential genetic repertoire of retroviruses. The goal of these studies is to test how physical properties of retroviral genomes affect viral replication success and how these factors contribute to the genetic make-up of retroviral populations. Towards these goals, we are evaluating the ability of structurally-aberrant retroviral RNAs to become encapsidated, to generate retroviral DNA, and to become integrated into host chromosomes in their DNA forms. We are also studying physical limitations of retroviral genome packaging: that is, whether RNA packaging is limited to a particular number of genomic RNAs or to a total mass of RNA, and whether alternative genome organization is compatible with replication. Retroviruses are known to be fairly tolerant of aberrant replication events, and when these rare events confer a selective advantage, they can result in the generation of retroviruses with altered properties. One important implication of this work is understanding retroviral population dynamics and how retroviruses change their pathogenic properties. Potential practical applications of these studies include that they may lead to the generation of retroviral vectors larger or in other ways better that those now in use.
We are also studying mechanistic details of the pathway of retroviral DNA synthesis. One approach to this is a melding of reverse transcriptase structural studies and genetics to examine interactions between reverse transcriptase and viral nucleic acids at key steps in retroviral DNA synthesis. An example is our genetic system to examine interactions between reverse transcriptase and genomic sequences critical to plus strand DNA initiation. Based in part on collaborative reverse transcriptase footprinting and crystallographic studies, we postulate that the specificity of both plus strand primer generation and primer utilization involve (possibly different) specific interactions between the DNA polymerase domain of reverse transcriptase and specific sequences in the so-called ppt region. We are examining which ppt alterations allow reverse transcriptase to discriminate among candidate ppt regions. By mapping critical nucleotides onto reverse transcriptase: nucleic acid models, we will identify and then selectively mutagenize candidate ppt recognition regions in reverse transcriptase. We can separate the effects of these enzyme: nucleic acid interactions on primer generation from those on primer utilization by using phenotypically mixed virions in which RNaseH and DNA polymerase activities are provided by different reverse transcriptase molecules. Such studies allow us to address fundamental questions about the specificity and mechanism of a simple replicative polymerase in a living system.