Background
Long-lived HIV-infected cells persist and undergo clonal expansion, preventing viral eradication during antiretroviral therapy. The majority of infected cells contain defective proviruses with mutations, deletions, or insertions. Defective proviruses are also generated by LTR-LTR recombination, excising the entire coding sequence, leaving only a solo LTR at the integration site. Little is known regarding solo HIV LTR formation and understanding mechanisms driving their formation will inform efforts to eradicate expression of HIV proteins.
Methods
Cell lines containing HIV proviruses (ACH-2, 8E5) and uninfected control (CEM) were maintained in vitro and sub-cloned. HIV DNA and RNA were quantified using multiplexed droplet digital PCR. Proviruses were recovered from cell lines using enrichment through biotin-labeled probes targeting HIV sequence and characterized by long-read sequencing. To screen host proteins required for the solo-LTR formation, we used a yeast genetic system that introduced a suicide gene in a LTR-retrotransposon. The LTR-LTR recombination leads to the knockout of the suicide gene, which allows us to investigate the frequency of LTR-LTR recombination.
Results
Using an enrichment and long read sequencing technique, we captured and characterized HIV-containing DNA fragments in latently HIV-1 infected cell lines. In 8E5 cells, we identified a full-length HIV provirus and a solo-LTR with the exact same integration site, demonstrating formation solo-LTR in vitro. PCR analyses with provirus-flanking primers revealed that the 8E5 cell line is a mixture of cells with either intact or solo-LTR proviruses. Analyses of multiple single cell clones containing full-length or solo-LTR HIV, including growth competition assays, revealed cells with solo-LTR exhibit growth advantage over cells containing full-length provirus. With yeast genetic assay we have revealed that the frequency of solo-LTR formation is approximately 10 events per million cells, which is consistent with the findings in the human endogenous retroviruses. Knockout of homologous recombination (HR) genes rad50 and rad52, orthologs of Human RAD50 and RAD52, substantially downregulated the frequency of LTR-LTR recombination.
Conclusions
Our group have demonstrated that solo-LTR HIV formation occurs in vitro. New cell lines generated in this study are now available to elucidate mechanisms to excise LTR coding sequences. With yeast system, we have identified HR plays crucial role in the formation of solo-LTR.