HIV DNA is heavily uracilated, which protects it from autointegration

Abstract

Human immune cells infected by HIV naturally contain high uracil content, and HIV reverse transcriptase (RT) does not distinguish between dUTP and dTTP. Many DNA viruses and retroviruses encode a dUTPase or uracil-DNA glycosylase (UNG) to counteract uracil incorporation. However, although HIV virions are thought to contain cellular UNG2, replication of HIV produced in cells lacking UNG activity does not appear to be impaired. Here we show that HIV reverse transcripts generated in primary human immune cells are heavily uracilated (>500 uracils per 10 kb HIV genome). We find that HIV DNA uracilation, rather than being dangerous, may promote the early phase of the viral life cycle. Shortly after reverse transcription, the ends of the HIV DNA are activated by the viral integrase (IN) in preparation for chromosomal insertion. However, the activated ends can attack the viral DNA itself in a suicidal side pathway, called autointegration. We find here that uracilation of target DNA inhibits the strand transfer of HIV DNA ends by IN, thereby inhibiting autointegration and facilitating chromosomal integration and viral replication. When uracilation is increased by incubating uracil-poor cells in the presence of increasing concentrations of dUTP or by infecting with virus that contains the cytosine deaminase APOBEC3G (A3G), the proportion of reverse transcripts that undergo suicidal autointegration decreases. Thus, HIV tolerates, or even benefits from, nonmutagenic uracil incorporation during reverse transcription in human immune cells.

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

“…As inferred from HIV-1 infections in nondividing cells (25), we found that HIV-1 readily incorporates dUTP during reverse transcription, and dUTP incorporation only produced a small kinetic delay in cDNA synthesis with no effect on the ultimate levels of viral cDNA. However, dUTP incorporation resulted in a sevenfold reduction in viral integration, and this reduction was completely dependent on the removal of uracils by nuclear hUNG2, leading to degradation of the viral genome.…”

“…3D). In fact, Yan et al (25) have proposed that uracilation of viral cDNA aids in integration by discouraging the competing and nonproductive autointegration reaction. Our results now uncover an additional consideration where nuclear hUNG2, if present at high enough levels, would efficiently degrade uracilated DNA.…”

“…We speculate that the reduction in viral protein is due to reduced expression from uracilated promoters or other important DNA elements. The absence of the 5-methyl group in uracil has the potential to alter DNA structure and dynamic features that might be important for recognition by transcription factors or other DNA binding proteins (23,25). For example, uracils localized in the origin of replication in HSV-1 were found to perturb the binding of the HSV-1 origin binding protein (27), and incorporated uracils in the cAMP response element (CRE) of rat stomatostatin gene led to a two-to fourfold decrease in binding of CRE binding proteins from HeLa extracts (26).…”

“…Finally, if the viral cDNA is uracilated to a sufficiently high density (12,25), it seems inevitable that the host nuclear UNG would excise the uracils, leading to its degradation once it entered the nucleus. There is little or no experimental data available to establish the importance of any of the above possible outcomes in human cells.…”

“…In turn, this may also compromise the terminal attachment site of the virus and inhibit integration (22). The presence of uracil in the viral cDNA may also induce structural changes that prevent correct interaction with proteins like integrase (23)(24)(25), and if uracil persists in the long terminal repeat (LTR) promoter of the integrated provirus, it may affect gene expression from the viral promoter (26,…”