The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein

Abstract

BackgroundDeoxyuridine 5′-triphosphate nucleotide-hydrolases (dUTPases) are essential for maintaining low intra-cellular dUTP/dTTP ratios. Therefore, many viruses encode this enzyme to prevent dUTP incorporation into their genomes instead of dTTP. Among the lentiviruses, the non-primate viruses express dUTPases. In bovine immunodeficiency virus (BIV), the putative dUTPase protein is only 74 residues-long, compared to ~130 residues in other lentiviruses.ResultsIn this study, the recombinant BIV dUTPase, as well as infectious wild-type (WT) BIV virions, were shown to lack any detectable dUTPase activity. Controls of recombinant dUTPase from equine infectious anemia virus (EIAV) or of EIAV virions showed substantial dUTPase activities. To assess the importance of the dUTPase to BIV replication, we have generated virions of WT BIV or BIV with mutations in the dUTPase gene. The two mutant viral dUTPases were the double mutant D48E/N57S (in the putative enzyme active site and its vicinity) and a deletion of 36 residues. In dividing Cf2Th cells and under conditions where the WT virus was infectious and generated progeny virions, both mutant viruses were defective, as no progeny viruses were generated. Analyses of the integrated viral cDNA showed that cells infected with the mutant virions carry in their genomic DNA levels of integrated BIV DNA that are comparable to those in WT BIV-infected cells.ConclusionsThe herby presented results show that the two BIV mutants with the modified dUTPase gene could infect cells, as viral cDNA was synthesized and integrated into the host cell DNA. However, no virions were generated by cells infected by these mutants. The most likely explanation is that either the integrated cDNA of the mutants is defective (due to potential multiple mutations, introduced during reverse-transcription) or that the original dUTPase mutations have led to severe blocks in viral replication at steps post integration. These results emphasize the importance of the dUTPase-related sequence to BIV replication, despite the lack of any detectable catalytic activity.Electronic supplementary materialThe online version of this article (doi:10.1186/1742-4690-11-60) contains supplementary material, which is available to authorized users.

dUTPase: the frequently overlooked enzyme encoded by many retroviruses

“…Some families of endogenous retroviruses, notably, HERVs K and ERVs share also dUTPase-related motifs with the five conserved segments [ 13 , 59 , 60 ]—see below. In contrast, the putative dUTPases of the non-primate lentivirus, bovine immunodeficiency virus (BIV) was recently shown by us to have a sequence with only a partial resemblance to these conserved motifs with no detectable enzymatic activity [ 61 ]—see below. The phylogenetic tree of most exogenous retroviral dUTPases, discussed below, is shown in Fig.…”

“…As in all non-primate lentiviruses, these genes are located between the RT and IN-encoding genes. However, in both cases the encoded polypeptide is substantially shorter than the ~130-residues protein of other lentiviruses, as it is only about 74 residues long [ 61 , 73 , 74 ]—see Fig. 5 .…”

“…5 . This truncated polypeptide lacks extensive parts of the five conserved motifs, characteristic of the homo-trimeric dUTPases, or the whole motifs [ 61 ]. As far as we know, no other dUTPase-related protein, including all viral, prokaryotic or eukaryotic enzymes is so small.…”

“…Still, it is highly likely that this dUTPase-related peptide has an important biological role, since it is conserved in both BIV and JDV [ 87 , 88 ]. Our recent study showed that recombinant wild-type BIV dUTPase and infectious wild-type BIV virions were both dUTPase-defective, as no detectable enzymatic activity could be shown [ 61 ]. To assess the importance of the dUTPase gene to BIV replication, we generated virions of wild-type BIV or BIV with mutations in this gene.…”

MicroRNA regulation of p21 and TASK1 cellular restriction-factors enhances HIV-1 infection

“…At 48 hours or 5 days post HeLa-CCR5 or JLTRG-R5 viral infection, supernatant samples were collected and viral expression was analyzed by RT-qPCR. Wild-type Moloney strain MLV (MMLV, 5µl) were added to the supernatant and served as the loading control for real-time PCR ( Voronin et al, 2014 ). Production of GFP by HIV-1-infected cells was measured daily for 14 or 21 consecutive days in a Synergy TM HT Multi-Detection Microplate Reader (Bio-Tek Instruments, USA), equipped with the following filter set: excitation, 485/20 nm; emission, 528/20 nm.…”