Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages

Adrian Oo,Dong-Hyun Kim,Raymond F Schinazi,Baek Kim

doi:10.1074/jbc.ra119.011466

Abstract

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain– and HD domain–containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase–mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

Highlights

Unlike activated CD4؉ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription
Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome observed with the infectious HIV-1 89.6 strain
The less effective dNTP pool elevation observed in dNtreated MDMs can be explained by the presence of SAMHD1 in these cells, which would hydrolyze dNTPs newly synthesized from the added dNs, unlike the complete SAMHD1 degradation observed in the Vpx-treated macrophages (Fig. S1)

Summary

Vpx reduces rNTP incorporation of lentiviruses in macrophage

Nonical rNTP misincorporation is an important contributing factor of cellular mutagenesis because the incorporated rNMP in dsDNA induces DNA polymerases pausing, which is known to be an error-prone event [14, 16, 17]. We previously demonstrated that HIV-1 frequently incorporates noncanonical rNTPs during viral reverse transcription in macrophages, but not in activated CD4ϩ T cells [23] because of a greater concentration discrepancy between rNTPs and dNTPs in macrophages compared with its actively dividing counterpart [24]. We tested whether SAMHD1-mediated dNTP depletion is responsible for the frequent incorporation of the noncanonical rNTPs during HIV-1 reverse transcription in macrophages and whether viral protein (X), an accessory protein expressed by HIV-2 and some SIV strains, can reduce rNTP incorporation by the virus RT via its SAMHD1-counteracting activity

Results

Discussion

Experimental procedures

Alkaline hydrolysis assay

Jurkat cell nuclear extract

Statistical analyses