Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages.
Abstract: The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full-length viral DNA by 24 hr postinfection. The level of integrated delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between viral DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from delta DU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into viral DNA.
Publication Date: 1995-07-10 PubMed ID: 7542416DOI: 10.1006/viro.1995.1347Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article explores how the incorporation of uracil into the DNA of the equine infectious anemia virus (EIAV) significantly reduces the virus’ ability to replicate in macrophages – a type of white blood cell. This reduction is linked to a deficiency in an enzyme called dUTPase, which is integral in preventing uracil incorporation.
Studying Various Forms of EIAV
- The researchers first created two different types of EIAV. One with a homogenous dUTPase deletion (delta DU EIAV) and another with a single amino acid substitution in dUTPase (DUD71E). Both of these mutated viruses displayed significantly reduced replication capabilities, reaching only 2% of the replication levels of the wild-type (unmodified) virus within seven days after infection.
The Role of dUTPase and DNA Synthesis
- The team sought to identify the blockade(s) in the replication process by examining the viruses’ DNA synthesis, integration and transcription stages. Although DNA synthesis was observably normal in the macrophages, the quantity of integrated mutated virus DNA was significantly lower (by 2-3 times) compared to the wild-type.
- However, the levels of full-length viral transcripts were observed to be lowered by a factor of more than 100. This indicated that the replication blockage predominantly occurs somewhere between the stages of viral DNA synthesis and transcription.
- Given that dUTPase helps to prevent uracil from being incorporated into freshly synthesized DNA, the researchers hypothesized that the mutated viruses’ DNA could be containing uracil due to the lack of dUTPase activity.
Proof of Uracil in Mutated Virus’ DNA
- Via in vitro assays, it was discovered that while wild-type viruses don’t use dUTP for their DNA synthesis, both the dUTPase-deficient variants synthesized DNA containing uracil.
- Following this, the presence of uracil in the DNA derived from macrophages infected by the mutant variants was also confirmed. This suggested that the dUTPase functions encoded by the virus are crucial to prevent uracil incorporation into the virus’ DNA during replication in macrophages.
Overall, the research offers valuable insights into the viral replication process, and potentially paves the way for new therapeutics to curb EIAV’s replication potential.
Cite This Article
APA
Steagall WK, Robek MD, Perry ST, Fuller FJ, Payne SL.
(1995).
Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages.
Virology, 210(2), 302-313.
https://doi.org/10.1006/viro.1995.1347 Publication
Researcher Affiliations
- Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
MeSH Terms
- Animals
- Base Sequence
- Binding Sites / genetics
- Cells, Cultured
- Cytopathogenic Effect, Viral
- DNA Glycosylases
- DNA Replication / physiology
- DNA, Viral / biosynthesis
- DNA, Viral / chemistry
- Horses
- Infectious Anemia Virus, Equine / enzymology
- Infectious Anemia Virus, Equine / physiology
- Macrophages / virology
- Molecular Sequence Data
- N-Glycosyl Hydrolases
- Point Mutation
- Pyrophosphatases / genetics
- Pyrophosphatases / metabolism
- Pyrophosphatases / physiology
- RNA-Directed DNA Polymerase
- Transcription, Genetic / genetics
- Uracil / analysis
- Uracil / metabolism
- Uracil-DNA Glycosidase
- Virus Integration
- Virus Replication / physiology
Grant Funding
- CA-59278 / NCI NIH HHS
- T32GM08056 / NIGMS NIH HHS
Citations
This article has been cited 25 times.- Munis AM. Gene Therapy Applications of Non-Human Lentiviral Vectors.. Viruses 2020 Sep 29;12(10).
- Meshesha M, Esadze A, Cui J, Churgulia N, Sahu SK, Stivers JT. Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages.. PLoS One 2020;15(7):e0235012.
- Li G, Wang C, Yang M, Cao L, Fu D, Liu X, Sun D, Chen C, Wang Y, Jia Z, Yang C, Guo Y, Rao Z. Structural Insight into African Swine Fever Virus dUTPase Reveals a Novel Folding Pattern in the dUTPase Family.. J Virol 2020 Jan 31;94(4).
- Schein CH. Polyglutamine Repeats in Viruses.. Mol Neurobiol 2019 May;56(5):3664-3675.
- Pollpeter D, Parsons M, Sobala AE, Coxhead S, Lang RD, Bruns AM, Papaioannou S, McDonnell JM, Apolonia L, Chowdhury JA, Horvath CM, Malim MH. Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G.. Nat Microbiol 2018 Feb;3(2):220-233.
- Hansen EC, Ransom M, Hesselberth JR, Hosmane NN, Capoferri AA, Bruner KM, Pollack RA, Zhang H, Drummond MB, Siliciano JM, Siliciano R, Stivers JT. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.. Elife 2016 Sep 20;5.
- Hizi A, Herzig E. dUTPase: the frequently overlooked enzyme encoded by many retroviruses.. Retrovirology 2015 Aug 12;12:70.
- Voronin N, Herzig E, Hizi A. The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein.. Retrovirology 2014 Aug 12;11:60.
- Weil AF, Ghosh D, Zhou Y, Seiple L, McMahon MA, Spivak AM, Siliciano RF, Stivers JT. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration.. Proc Natl Acad Sci U S A 2013 Feb 5;110(6):E448-57.
- Qi X, Wang X, Wang S, Lin Y, Jiang C, Ma J, Zhao L, Lv X, Shen R, Wang F, Kong X, Su Z, Zhou J. Genomic analysis of an effective lentiviral vaccine-attenuated equine infectious anemia virus vaccine EIAV FDDV13.. Virus Genes 2010 Aug;41(1):86-98.
- Elder JH, Lin YC, Fink E, Grant CK. Feline immunodeficiency virus (FIV) as a model for study of lentivirus infections: parallels with HIV.. Curr HIV Res 2010 Jan;8(1):73-80.
- De Silva FS, Moss B. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice.. Virol J 2008 Dec 2;5:145.
- Sire J, Quérat G, Esnault C, Priet S. Uracil within DNA: an actor of antiviral immunity.. Retrovirology 2008 Jun 5;5:45.
- Elder JH, Sundstrom M, de Rozieres S, de Parseval A, Grant CK, Lin YC. Molecular mechanisms of FIV infection.. Vet Immunol Immunopathol 2008 May 15;123(1-2):3-13.
- Kaiser SM, Emerman M. Uracil DNA glycosylase is dispensable for human immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase Apobec3G.. J Virol 2006 Jan;80(2):875-82.
- Maury W, Wright PJ, Bradley S. Characterization of a cytolytic strain of equine infectious anemia virus.. J Virol 2003 Feb;77(4):2385-99.
- Miller RJ, Cairns JS, Bridges S, Sarver N. Human immunodeficiency virus and AIDS: insights from animal lentiviruses.. J Virol 2000 Aug;74(16):7187-95.
- Mansky LM, Preveral S, Selig L, Benarous R, Benichou S. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate.. J Virol 2000 Aug;74(15):7039-47.
- Abergel C, Robertson DL, Claverie JM. "Hidden" dUTPase sequence in human immunodeficiency virus type 1 gp120.. J Virol 1999 Jan;73(1):751-3.
- Pétursson G, Turelli P, Matthíasdóttir S, Georgsson G, Andrésson OS, Torsteinsdóttir S, Vigne R, Andrésdóttir V, Gunnarsson E, Agnarsdóttir G, Quérat G. Visna virus dUTPase is dispensable for neuropathogenicity.. J Virol 1998 Feb;72(2):1657-61.
- Maury W, Perryman S, Oaks JL, Seid BK, Crawford T, McGuire T, Carpenter S. Localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus.. J Virol 1997 Jul;71(7):4929-37.
- Selig L, Benichou S, Rogel ME, Wu LI, Vodicka MA, Sire J, Benarous R, Emerman M. Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.. J Virol 1997 Jun;71(6):4842-6.
- Turelli P, Guiguen F, Mornex JF, Vigne R, Quérat G. dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.. J Virol 1997 Jun;71(6):4522-30.
- Weiss RS, Lee SS, Prasad BV, Javier RT. Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.. J Virol 1997 Mar;71(3):1857-70.
- Prasad GS, Stura EA, McRee DE, Laco GS, Hasselkus-Light C, Elder JH, Stout CD. Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus.. Protein Sci 1996 Dec;5(12):2429-37.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
