Search

Abstract Uracil is a natural base of RNA but may appear in DNA through two different pathways including cytosine deamination or misincorporation of deoxyuridine 5'-triphosphate nucleotide (dUTP) during DNA replication and constitutes one of the most frequent DNA lesions. In cellular organisms, such lesions are faithfully cleared out through several universal DNA repair mechanisms, thus preventing genome injury. However, several recent studies have brought some pieces of evidence that introduction of uracil bases in viral genomic DNA intermediates during genome replication might be a way of innate immune defence against some viruses. As part of countermeasures, numerous viruses have developed powerful strategies to prevent emergence of uracilated viral genomes and/or to eliminate uracils already incorporated into DNA. This review will present the current knowledge about the cellular and viral countermeasures against uracils in DNA and the implications of these uracils as weapons against viruses.

Alphaviruses are arthropod-borne viruses of public health concern. To date no efficient vaccine nor antivirals are available for safe human use. During viral replication the nonstructural protein 1 (nsP1) catalyzes capping of genomic and subgenomic RNAs. The capping reaction is unique to the Alphavirus genus. The whole three-step process follows a particular order: (i) transfer of a methyl group from S-adenosyl methionine (SAM) onto a GTP forming m7 GTP; (ii) guanylylation of the enzyme to form a m7 GMP-nsP1adduct; (iii) transfer of m7 GMP onto 5'-diphosphate RNA to yield capped RNA. Specificities of these reactions designate nsP1 as a promising target for antiviral drug development. In the current study we performed a mutational analysis on two nsP1 positions associated with Sindbis virus (SINV) ribavirin resistance in the Venezuelan equine encephalitis virus (VEEV) context through reverse genetics correlated to enzyme assays using purified recombinant VEEV nsP1 proteins. The results demonstrate that the targeted positions are strongly associated to the regulation of the capping reaction by increasing the affinity between GTP and nsP1. Data also show that in VEEV the S21A substitution, naturally occurring in Chikungunya virus (CHIKV), is a hallmark of ribavirin susceptibility. These findings uncover the specific mechanistic contributions of these residues to nsp1-mediated methyl-transfer and guanylylation reactions., (Copyright © 2020. Published by Elsevier B.V.)

Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVB res ) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve ∼18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVB res was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development., (Copyright © 2020 American Society for Microbiology.)

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m 7 GMP releasing pyrophosphate (GT reaction) and the m 7 GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m 7 GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC 50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bunyaviridae family is the largest and most diverse family of RNA viruses. It has more than 350 members divided into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. They are present in the five continents, causing recurrent epidemics, epizootics, and considerable agricultural loss. The genome of bunyaviruses is divided into three segments of negative single-stranded RNA according to their relative size: L (Large), M (Medium) and S (Small) segment. Bunyaviridae RNA-dependent RNA polymerase (RdRp) is encoded by the L segment, and is in charge of the replication and transcription of the viral RNA in the cytoplasm of the infected cell. Viral RdRps share a characteristic right hand-like structure with three subdomains: finger, palm, and thumb subdomains that define the formation of the catalytic cavity. In addition to the N-terminal endonuclease domain, eight conserved motifs (A-H) have been identified in the RdRp of Bunyaviridae. In this review, we have summarized the recent insights from the structural and functional studies of RdRp to understand the roles of different motifs shared by RdRps, the mechanism of viral RNA replication, genome segment packaging by the nucleoprotein, cap-snatching, mRNA transcription, and other RNA mechanisms of bunyaviruses.

Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF ® ) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC 50 determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases., (Copyright © 2017. Published by Elsevier B.V.)

Ebola virus (EBOV) haemorrhagic fever remains a threat to global public health with an urgent need for an effective treatment. In order to achieve these goals, access to non-human primate (NHP) laboratory models is an essential requirement. Here, we present the first NHP-EBOV laboratory model readily available to the European scientific community, based on infection of Mauritian cynomolgus macaques using a Central-African EBOV strain and increasing virus challenge dose (10, 100, or 1000 focus forming units per animal). The outcome of these experiments was assessed using clinical, hematological, and biochemical criteria. All challenge doses resulted in fatal infections within 8-11 days. Symptoms appeared from day 5 after infection onwards and disease progression was slower than in previous reports based on Asian cynomolgus macaques. Thus, our model resembled human disease more closely than previous models (onset of symptoms estimated 2-21 days after infection) extending the period of time available for therapeutic intervention. To establish the dynamics of virus genome variation, the study included the first detailed analysis of major and minor genomic EBOV variants during the course of the disease. Major variants were scarce and the population of minor variants was shaped by selective pressure similar to genomic mutations observed in Nature. This primate model provides a robust baseline for future genomic studies in the context of therapeutic methods for treating Ebola virus-infected patients., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: T-705, also known as favipiravir, is a small-molecule inhibitor that is currently in clinical development for the treatment of influenza virus infections. This molecule also inhibits the replication of a broad spectrum of other RNA viruses. The objective of this study was to investigate the antiviral effect of favipiravir on chikungunya virus (CHIKV) replication and to contribute to unravelling the molecular mechanism of action against this virus., Methods: The anti-CHIKV effect of favipiravir was examined in cell culture and in a mouse model of lethal infection. A five-step protocol was used to select for CHIKV variants with reduced susceptibility to favipiravir. The resistant phenotype was confirmed in cell culture and the whole genome was sequenced. The identified mutations were reverse-engineered into an infectious clone to confirm their impact on the antiviral efficacy of favipiravir., Results: Favipiravir inhibits the replication of laboratory strains and clinical isolates of CHIKV, as well as of a panel of other alphaviruses. Several favipiravir-resistant CHIKV variants were independently selected and all of them in particular acquired the unique K291R mutation in the RNA-dependent RNA polymerase (RdRp). Reverse-engineering of this K291R mutation into an infectious clone of CHIKV confirmed the link between the mutant genotype and the resistant phenotype. Interestingly, this particular lysine is also highly conserved in the RdRp of positive-stranded RNA viruses in general., Conclusions: This study provides an important insight into the precise molecular mechanism by which favipiravir exerts its antiviral activity against (alpha)viruses, which may be of help in designing other potent broad-spectrum antivirals., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Flaviviruses are single-stranded positive RNA viruses that replicate through double stranded RNA (dsRNA) intermediates. These dsRNA may be recognized as pathogen-associated molecular patterns by cellular receptors including membrane-bound Toll-like receptor 3 (TLR3) and cytosolic helicases RIG-I and MDA5. dsRNA stimulation results in signaling cascades converging to activation of interferon (IFN) regulatory factor 3 (IRF3) and to transcriptional activation of several interferon stimulated genes, including IFNss and inflammatory cytokines. There are conflicting reports concerning the ability of West Nile virus to counteract TLR3 signaling. In our analyses, transiently or stably expressed NS1 proteins from two West Nile viruses, two dengue 2 viruses and a yellow fever virus failed to inhibit TLR3 signaling in two different mammalian cell lines. Moreover, using siRNA inhibiting the helicase signalization pathway, we show that viral infection did not impede TLR3 responses to poly(I:C). We conclude that NS1 proteins from distinct mosquito-borne flaviviruses do not inhibit TLR3 signaling., (2010 Elsevier Inc. All rights reserved.)

Uracil in DNA is a deleterious event that may arise either by cytosine deamination or misincorporation of dUTP. Consequently, cells from all free-living organisms have developed strategies to protect their genome against the presence of uracils, by using uracil DNA glycosylase (UNG) and deoxyuridine triphosphatase (dUTPase) enzymatic activities. In the viral kingdom, some (namely poxviruses and herpesviruses) but not all of the DNA viruses encode their own UNG and dUTPase to control uracilation of their genome. Some retroviruses, which are RNA viruses using DNA as an intermediate of replication, also encode dUTPase. Surprisingly, though most of nonprimate lentiviruses encode dUTPase, primate lentiviruses such as HIV-1, HIV-2 or SIV do not. Because these latter viruses also replicate in nondividing cells where the dUTP/dTTP ratio is high, it is probable that they have found other ways to fight against the emergence of uracilated-viral transcripts. Indeed, recent studies showed that HIV-1 efficiently controls both the cytosine deamination and the dUTP misincorporation. The viral Vif protein acts in preventing the packaging into viral particles of the host-derived cytosine deaminase APOBEC3G enzyme, while the viral integrase domain of the Gag-Pol precursor mediates the packaging of the host-derived uracil DNA glycosylase UNG2 enzyme. In the absence of Vif or UNG2, HIV-1 viral transcripts are heavily charged in uracil bases leading to inactivation of the virus.

Uracilation of DNA represents a constant threat to the survival of many organisms including viruses. Uracil may appear in DNA either by cytosine deamination or by misincorporation of dUTP. The HIV-1-encoded Vif protein controls cytosine deamination by preventing the incorporation of host-derived APOBEC3G cytidine deaminase into viral particles. Here, we show that the host-derived uracil DNA glycosylase UNG2 enzyme, which is recruited into viral particles by the HIV-1-encoded integrase domain, is essential to the viral life cycle. We demonstrate that virion-associated UNG2 catalytic activity can be replaced by the packaging of heterologous dUTPase into virion, indicating that UNG2 acts to counteract dUTP misincorporation in the viral genome. Therefore, HIV-1 prevents incorporation of dUTP in viral cDNA by UNG2-mediated uracil excision followed by a dNTP-dependent, reverse transcriptase-mediated endonucleolytic cleavage and finally by strand-displacement polymerization. Our findings indicate that pharmacologic strategies aimed toward blocking UNG2 packaging should be explored as potential HIV/AIDS therapeutics.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for integration of viral DNA into host cell chromatin. We have reported previously (Priet, S., Navarro, J. M., Gros, N., Querat, G., and Sire, J. (2003) J. Biol. Chem. 278, 4566-4571) that IN also plays a role in the packaging of the host uracil DNA glycosylase UNG2 into viral particles and that the region of IN encompassing residues 170-180 was responsible for the interaction with UNG2 and for its packaging into virions. In this work, we aimed to investigate the replication of HIV-1 viruses rendered deficient in virion-associated UNG2 by single or double point mutations in the region 170-180 of IN. We show that the L172A/K173A IN mutant virus was deficient for UNG2 packaging and was defective for replication because of a blockage at the stage of proviral DNA integration in host cell DNA. In vitro assays using long term repeat mimics, however, demonstrate that the L172A/K173A IN mutant was catalytically active. Moreover, trans-complementation experiments show that the viral propagation of L172A/K173A viruses could be rescued by the overexpression of Vpr.L172A/K173A IN fusion protein in a dose-dependent manner and that this rescue is independent of UNG2 packaging. Altogether, our data indicate that L172A/K173A mutations of IN induce a subtle defect in the function of IN, which nevertheless dramatically impairs viral replication. Unexpectedly, this blockage of replication could be overcome by forcing the packaging of higher amounts of this same mutated integrase. This is the first study reporting that blockage of the integration process of HIV-1 provirus carrying a mutation of IN could be alleviated by increasing amounts of IN even carrying the same mutations.

We have previously reported that the host uracil DNA glycosylase UNG2 enzyme is incorporated into HIV-1 virions via a specific association with the viral integrase (IN) domain of Gag-Pol precursor. In this study, we investigated whether UNG2 was packaged into two phylogenetically closely related primate lentiviruses, HIV-2(ROD) and SIV(MAC239). We demonstrated by GST-pull-down and coprecipitation assays that INs from HIV-1, HIV-2(ROD), and SIV(MAC239) associated with UNG2, although the interaction of UNG2 with HIV-2(ROD) IN and SIV(MAC239) IN was less strong than with HIV-1 IN. We then showed by Western blotting that highly purified HIV-2 and SIV(MAC) viral particles did not incorporate host UNG2, contrasting with the presence of UNG2 in HIV-1 viral particles. Finally, we showed that HIV-1/SIV chimeric viruses in which residues 6 to 202 of HIV-1 IN were replaced by the SIV counterpart were impaired for packaging of UNG2, indicating that the incorporation of host UNG2 into viral particles is the hallmark of the HIV-1 strain. Moreover, we found that HIV-1/SIV IN chimeric viruses were deficient for viral propagation.

The biological form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer consisting of two polypeptides, p66 and p51, which have identical N-termini. The p51 polypeptide is generated by action of viral protease cleaving the p66 polypeptide between residues Phe440 and Tyr441. Dimerization has been mostly studied using bacterially purified RT bearing amino acid changes in either subunit, but not in the context of HIV-1 particles. We introduced changes of conserved amino acid residues 430-438 into the protease-sensitive subdomain of the p66 subunit and analyzed the reverse transcriptase processing and function using purified variants and their corresponding HIV-1 recombinant clones. Our mutational analysis shows that the conserved Glu438 residue is critical for proper heterodimerization and function of virion-associated RT, but not of bacterially expressed RT. In contrast, the conserved Glu430, Glu432, and Pro433 residues are not important for dimerization of virion-associated RT. The network of interactions made by the Glu438 carboxyl group with neighboring residues is critical to protect the Phe440-Tyr441 from cleavage in the context of the p66/p51 heterodimer and may explain why the p66/p51 is not processed further to p51/p51.

The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.

In the course of attempts to grow the jaagsiekte retrovirus in cell culture, a typical lentivirus was isolated for the first time in South Africa from adenomatous lungs. Morphologically the virus could not be distinguished from other lentiviruses, but serologically it was shown to be more closely related to visna virus than to caprine arthritis-encephalitis virus. However, a preliminary restriction enzyme analysis of the linear proviral DNA of this new lentivirus (SA-DMVV) revealed that it is significantly district from visna virus and CAEV and therefore may represent a third type of lentivirus. Antibodies to the virus were demonstrated in a number of sheep in various parts of the country, but a direct link to a disease condition was not found. Attempts to produce lung lesions by intratracheal injection of the virus have been unsuccessful to date but a transient arthritis was produced by intraarticular inoculation. Viral replication seems to be enhanced in jaagsiekte lungs.

Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.