Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

Abstract

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.

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

“…A requirement for hUNG2 in HIV-1 infection has been debated for >15 y. hUNG2 is widely reported to be packaged into HIV-1 particles in producer cells through interactions of its N-terminal domain with the HIV-1 accessory protein Vpr (38)(39)(40)48) [although a packaging interaction with viral integrase has also been reported (49)]. The proposed roles for packaged and nuclear hUNG2 in the viral life cycle have been diverse: (i) the virally packaged enzyme serves to nonenzymatically or enzymatically suppress mutations in the viral genome upon infection of macrophages by positively influencing reverse transcription (38,39,41,50), (ii) the virally packaged UNG2 is completely dispensable for HIV-1 replication of cells with low dUTP levels (45), (iii) virion-associated hUNG2 degrades APOBEC3-edited HIV-1 DNA (46), (iv) virionassociated and nuclear hUNG2 play a selective role in enhancing infection of primary lymphocytes by R5 tropic, but not R4 tropic or pseudotyped HIV-1 (13). This latter role for hUNG2 appears to be entirely independent of dUTP levels, and in addition, is unrelated to our observations because we use a VSV-G pseudotyped HIV-1.…”

“…There are several reports that viral protein R (Vpr) recruits UNG2 into HIV-1 particles in producer cells, although the implication of this recruitment is controversial (38)(39)(40). To ascertain whether our findings were influenced by the presence of virally packaged hUNG2, we repeated the infection experiments with viruses that encode two defective forms of Vpr.…”

Uracil-Directed Ligand Tethering: An Efficient Strategy for Uracil DNA Glycosylase (UNG) Inhibitor Development

“…This substrate was easier to synthesize and purify than an all DNA hairpin, and unlike the double stranded DNA substrate, does not require MgCl 2 to achieve minimum fluorescence. To a 96-well plate was added 5 μL compound in DMSO, followed by 75 μL PEG-U9 hairpin in reaction buffer (20 …”

Effects of cell cycle status on early events in retroviral replication

“…We focus on those cell cycle dependencies that can lead to a partial or complete restriction of early events, as well as viral mechanisms that help overcome these restrictions. Although we have highlighted early events in retroviral replication, it should be noted that a broad array of retroviral gene products, including oncoproteins [Coffin et al, 1997;Flint, 2004] and accessory proteins [Amini et al, 2004;Chen et al, 2004;Goh et al, 2004] can also influence the cell cycle in many ways.…”

“…The HIV-1 accessory protein, Vpr interacts with a cellular uracil-DNA glycosylase 2 (UNG2) and mediates its assembly into viral particles [Chen et al, 2002]. The assembled cellular UNG2 may facilitate repair of dUMP residues that have been incorporated into viral DNA, thereby acting as a functional equivalent of the retrovirus-encoded dUTPases [Chen et al, 2004]. These findings highlight the importance of accessory functions that affect viral DNA synthesis to facilitate viral propagation in noncycling cells.…”