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

Nickolay Voronin,Eytan Herzig,Amnon Hizi

doi:10.1186/1742-4690-11-60

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.

Highlights

Deoxyuridine 5′-triphosphate nucleotide-hydrolases are essential for maintaining low intra-cellular dUTP/dTTP ratios
What is the mode by which the mutations introduced into the dUTPase gene have led to blocking viral replication? The results presented in Figure 9 show that the extent of viral cDNA synthesis and the subsequent cDNA integration into the cellular DNA were not affected by the dUTPase mutations, indicating that the early steps of the retroviral infection were quite normal
Non-primate lentiviruses express a dUTPase enzyme that is important for viral growth in non-replicating cells

Summary

Introduction

Deoxyuridine 5′-triphosphate nucleotide-hydrolases (dUTPases) are essential for maintaining low intra-cellular dUTP/dTTP ratios. Cellular deoxyuridine 5′-triphosphate nucleotide-hydrolases (dUTPases) have an essential role in maintaining low cellular dUTP over dTTP ratios. This is done by catalyzing the hydrolysis of dUTP into two products, the dTTP precursor-dUMP, and pyrophosphate (PPi) [1,2,3]. While synthesizing DNA, RT is capable of incorporating dUTP instead of dTTP into the nascent DNA strands [7,8] This may explain why retroviral dUTPases are associated with increasing viral replication efficiency and a better fidelity of DNA synthesis, prevent deleterious mutations [9]. Most dUTPases (including the retroviral ones) are homotrimers with five conserved sequence motifs [15], see Figure 1A

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