Understanding Retroviral Reverse Transcription, Recombination, and Replication

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

During replication, HIV-1 converts its packaged dimeric RNA genomes into DNA and generates one provirus in an infection event. In this process, HIV-1 needs to preserve its genetic information while the host innate immune response attempts to abolish the generation of proviruses capable of producing infectious progeny. HIV-1 must protect its genome during virus infection while navigating through the host cell. Dr. Vinay Pathak (HIV DRP) and colleague has shown that HIV-1 cores that retained 94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site 1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. We have assisted Dr. Pathak's section to address this question. Using a GFP content marker, we found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker on average of 1 to 3 min before loss of capsid-associated host factor (mRuby-CPSF6). These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration. HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Additionally, we have assisted Dr. Pathak's section to investigate the intranuclear positions of transcriptionally active HIV-1 proviruses. We found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of proviral integration are dynamic. Additionally, we observed that HIV-1 cores were localized to nuclear speckles shortly after nuclear import, but transcriptionally active proviruses were located adjacent to nuclear speckles. Overall, these studies provide insights into HIV-1 integration site selection and their effect on transcription activities. We have also assisted Dr. Mary Kearney's group (HIV DRP) to establish a bioinformatic pipeline to define the intactness of HIV-1 proviral genomes identified in sequencing of clinical samples.