Viral Polymerase Research Articles

The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Infectious bronchitis virus: an overview of the "chicken coronavirus".

Infectious bronchitis virus (IBV) is a highly contagious avian Gammacoronavirus that affects mainly chickens (Gallus gallus) but can circulate in other avian species. IBV constitutes a significant threat to the poultry industry, causing reduced egg yield, growth and mortality levels that can vary in impact. The virus can be transmitted horizontally by inhalation or direct/indirect contact with infected birds or contaminated fomites, vehicles, farm personnel and litter (Figure 1). The error-prone viral polymerase and recombination mechanisms mean diverse viral population results, with multiple genotypes, serotypes, pathotypes and protectotypes. This significantly complicates control and mitigation strategies based on vigilance in biosecurity and the deployment of vaccination.

Small Molecule Drugs Targeting Viral Polymerases.

Small molecules that specifically target viral polymerases-crucial enzymes governing viral genome transcription and replication-play a pivotal role in combating viral infections. Presently, approved polymerase inhibitors cover nine human viruses, spanning both DNA and RNA viruses. This review provides a comprehensive analysis of these licensed drugs, encompassing nucleoside/nucleotide inhibitors (NIs), non-nucleoside inhibitors (NNIs), and mutagenic agents. For each compound, we describe the specific targeted virus and related polymerase enzyme, the mechanism of action, and the relevant bioactivity data. This wealth of information serves as a valuable resource for researchers actively engaged in antiviral drug discovery efforts, offering a complete overview of established strategies as well as insights for shaping the development of next-generation antiviral therapeutics.

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Sodium Polyoxotungstate Inhibits the Replication of Influenza Virus by Blocking the Nuclear Import of vRNP.

Both pandemic and seasonal influenza are major health concerns, causing significant mortality and morbidity. Current influenza drugs primarily target viral neuraminidase and RNA polymerase, which are prone to drug resistance. Polyoxometalates (POMs) are metal cation clusters bridged by oxide anions. They have exhibited potent anti-tumor, antiviral, and antibacterial effects. They have remarkable activity against various DNA and RNA viruses, including human immunodeficiency virus, herpes simplex virus, hepatitis B and C viruses, dengue virus, and influenza virus. In this study, we have identified sodium polyoxotungstate (POM-1) from an ion channel inhibitor library. In vitro, POM-1 has been demonstrated to have potent antiviral activity against H1N1, H3N2, and oseltamivir-resistant H1N1 strains. POM-1 can cause virion aggregation during adsorption, as well as endocytosis. However, the aggregation is reversible; it does not interfere with virus adsorption and endocytosis. Our results suggest that POM-1 exerts its antiviral activity by inhibiting the nuclear import of viral ribonucleoprotein (vRNP). This distinct mechanism of action, combined with its wide range of efficacy, positions POM-1 as a promising therapeutic candidate for influenza treatment and warrants further investigation.

Structures of the mumps virus polymerase complex via cryo-electron microscopy

The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L–P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.

Respiratory syncytial virus in pediatric patients admitted to a tertiary center in Amman: clinical characteristics, and age-related patterns

BackgroundRespiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infections, particularly in infants and young children during winter. We aimed to study the demographics and clinical characteristics of RSV infections and age-related patterns.MethodsThis retrospective study evaluated pediatric respiratory syncytial virus (RSV) infections conducted in Jordan from September 2021 to March 2022. Patients under the age of five who had viral polymerase chain reaction results showing RSV infection from nasopharyngeal aspiration were included. In addition, demographic information, medical history, and clinical data were gathered. These included comorbidities, outcomes, length of stay, ICU hospitalization, use of antibiotics, and oxygen supplementation.ResultsA total of 199 patients were included. Most patients were males (56.8%) and less than one year (43.7%). Children aged between 1 and 2 years presented with more shortness of breath (90.1%) than infants and children more than two years (66.7% and 87%, respectively) (p < 0.001). Older children (> 2 years) were significantly more likely to use antibiotics and have ICU admission than younger children ≤ 2 years (p = 0.045 and 0.018, respectively). There was no relationship between age groups, recurrent hospitalization, previous RSV infection, oxygen therapy, coinfection, and hospitalization duration. The respiratory rate was higher among patients with co-infection (p = 0.031).ConclusionThe current study provides information on the demographics and clinical characteristics of RSV infections. These findings contribute to a nuanced understanding of RSV infections in the specified population, emphasizing age-specific variations and clinical implications for better management strategies.

Exploration of influenza A virus PA protein-associated cellular proteins discloses its impact on mitochondrial function

Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LC‒MS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.

HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Ocular manifestations of Monkeypox virus (MPXV) infection with viral persistence in ocular samples: A case series

ObjectiveWe describe the clinical presentation and ocular viral dynamics in patients with Monkeypox virus-related ophthalmic disease (MPXROD). MethodsIn this case series, we investigated five consecutive patients with confirmed mpox, diagnosed through a positive Monkeypox virus (MPXV) Polymerase Chain Reaction (PCR) test and presenting with ocular symptoms. They were referred from the Reference Center for Sexually Transmitted Infections in São Paulo (CRT) to the Uveitis Sector at the Federal University of São Paulo, between August and December 2022. We performed PCR testing on ocular samples and culture supernatants for MPXV in all patients. Viral sequencing was conducted in one of the cases. ResultsReplicating MPXV was identified in at least one ocular sample of all patients, between day 31 and day 145 after the onset of skin lesions. All patients presented with keratitis, 3 with uveitis (60%) and two exhibited hypopyon (40%). The onset of ocular symptoms occurred at a mean of 21.2 days after the appearance of the first skin lesion and persisted, on average, for 61,.6 days, with a worsening trend observed until the initiation of tecovirimat treatment. Tecovirimat treatment was administered to all patients, with initiation occurring between 31 and 145 days after the onset of skin lesions. MPXV genome sequencing of an isolate from one patient classified it as belonging to lineage B1 in clade IIb. ConclusionThis study reveals a late onset and persistence of sight threatening ocular disease, along with potential viral infectivity even after systemic resolution in mpox cases. These findings highlight the risk of ongoing transmission from individuals with prolonged ocular manifestations, particularly through ocular discharge.

Structure-Based Discovery of Allosteric Inhibitors Targeting a New Druggable Site in the Respiratory Syncytial Virus Polymerase.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infections for which effective treatment options remain limited. Herein, we employed a computational structure-based design strategy aimed at identifying potential targets for a new class of allosteric inhibitors. Our investigation led to the discovery of a previously undisclosed allosteric binding site within the RSV polymerase, the large (L) protein. This discovery was achieved through a combination of virtual screening and molecular dynamics simulations. Subsequently, we identified two inhibitors, 6a and 10b, which both exhibited promising antiviral activity in the low micromolar range. Resistance profiling revealed a distinctive pattern in how RSV evaded treatment with this class of inhibitors. This pattern strongly suggested that this class of small molecules was targeting a new binding site in the RSV L protein, aligning with the computational predictions made in our study. This study paves the way for the development of more potent inhibitors for combating RSV infections by targeting a new druggable pocket within the RdRp which does not overlap with previously known resistance sites.

Structural and functional insights in flavivirus NS5 proteins gained by the structure of Ntaya virus polymerase and methyltransferase

Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5' RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.

Computational analysis of antiviral drugs using topological descriptors

Many health challenges are attributed to viral infections, which represent significant concerns in public health. Among these infections, diseases such as herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella-zoster virus (VZV) infections have garnered attention due to their prevalence and impact on human health. There are specific antiviral medications available for the treatment of these viral infections. Drugs like Cidofovir, Valacyclovir, and Acyclovir are commonly prescribed. These antiviral drugs are known for their efficacy against herpesviruses and related viral infections, leveraging their ability to inhibit viral DNA polymerase. A molecular descriptor is a numerical value that correlates with specific physicochemical properties of a molecular graph. This article explores the calculation of distance-based topological descriptors, including the Trinajstic, Mostar, Szeged, and PI descriptors for the aforementioned antiviral drugs. These descriptors provide insights into these drugs’ structural and physicochemical characteristics, aiding in understanding their mechanism of action and the development of new therapeutic agents.

Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19.

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

The artificial production of viral-like particles in Nicotiana benthamiana suggests the pro-assembly role of the Cannabis cryptic virus RdRP

AbstractThe partitivirids feature an icosahedral protein coating accommodating both their dsRNA genome and RNA-dependent RNA polymerase (RdRP). This signifies that transcription and replication activities of the viral polymerase occur within the capsid, emphasizing that the viral cycle relies on polymerase incorporation. Particles lacking RdRP are defective and hence non-infectious. Encapsidation and replication are intricately linked for dsRNA viruses, to the extent that, for many of these, such as the cystovirids, the RdRP serves a dual role as a transcriptase/replicase and a pro-assembly factor, ensuring structural stability and overall capsid integrity. This work investigates if RdRP has a similar role within the capsid of Cannabis cryptic virus (CanCV), a betapartitivirus affecting Cannabis sativa. Utilizing reverse genetics in Nicotiana benthamiana, we conclusively established that RdRP expression is indispensable for CanCV’s virus-like particle formation. This study enhances our understanding of CanCV encapsidation, with RdRP serving a pivotal role as a pro-assembly factor. These preliminary findings contribute to the knowledge of viral assembly within the Partitiviridae family.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

The varicella-zoster virus ORF16 protein promotes both the nuclear transport and the protein abundance of the viral DNA polymerase subunit ORF28

Although all herpesviruses utilize a highly conserved replication machinery to amplify their viral genomes, different members may have unique strategies to modulate the assembly of their replication components. Herein, we characterize the subcellular localization of seven essential replication proteins of varicella-zoster virus (VZV) and show that several viral replication enzymes such as the DNA polymerase subunit ORF28, when expressed alone, are localized in the cytoplasm. The nuclear import of ORF28 can be mediated by the viral DNA polymerase processivity factor ORF16. Besides, ORF16 could markedly enhance the protein abundance of ORF28. Noteworthily, an ORF16 mutant that is defective in nuclear transport still retained the ability to enhance ORF28 abundance. The low abundance of ORF28 in transfected cells was due to its rapid degradation mediated by the ubiquitin-proteasome system. We additionally reveal that radicicol, an inhibitor of the chaperone Hsp90, could disrupt the interaction between ORF16 and ORF28, thereby affecting the nuclear entry and protein abundance of ORF28. Collectively, our findings imply that the cytoplasmic retention and rapid degradation of ORF28 may be a key regulatory mechanism for VZV to prevent untimely viral DNA replication, and suggest that Hsp90 is required for the interaction between ORF16 and ORF28.

Reactivation of Multidrug-Resistant HSV-1 in a Post-Allogenic Hematopoietic Stem Cell Transplant Patient: Dynamic Detection of the Rare A605V Mutation by Next-Generation Sequencing.

We present an immunocompromised patient with a multiresistant herpes simplex virus-1 reactivation with a rare mutation (A605V) in the viral DNA polymerase gene. Next-generation sequencing suggests the presence of multiple drug-resistant strains before treatment and altered ratios during treatment, affecting the clinical response to aciclovir and foscarnet.