Viral RNA Polymerase Research Articles

Biomarkers in Detection of Hepatitis C Virus Infection.

The hepatitis C virus (HCV) infection affects 58 million people worldwide. In the United States, the incidence rate of acute hepatitis C has doubled since 2014; during 2021, this increased to 5% from 2020. Acute hepatitis C is defined by any symptom of acute viral hepatitis plus either jaundice or elevated serum alanine aminotransferase (ALT) activity with the detection of HCV RNA, the anti-HCV antibody, or hepatitis C virus antigen(s). However, most patients with acute infection are asymptomatic. In addition, ALT activity and HCV RNA levels can fluctuate, and a delayed detection of the anti-HCV antibody can occur among some immunocompromised persons with HCV infection. The detection of specific biomarkers can be of great value in the early detection of HCV infection at an asymptomatic stage. The high rate of HCV replication (which is approximately 1010 to 1012 virions per day) and the lack of proofreading by the viral RNA polymerase leads to enormous genetic diversity, creating a major challenge for the host immune response. This broad genetic diversity contributes to the likelihood of developing chronic infection, thus leading to the development of cirrhosis and liver cancer. Direct-acting antiviral (DAA) therapies for HCV infection are highly effective with a cure rate of up to 99%. At the same time, many patients with HCV infection are unaware of their infection status because of the mostly asymptomatic nature of hepatitis C, so they remain undiagnosed until the liver damage has advanced. Molecular mechanisms induced by HCV have been intensely investigated to find biomarkers for diagnosing the acute and chronic phases of the infection. However, there are no clinically verified biomarkers for patients with hepatitis C. In this review, we discuss the biomarkers that can differentiate acute from chronic hepatitis C, and we summarize the current state of the literature on the useful biomarkers that are detectable during acute and chronic HCV infection, liver fibrosis/cirrhosis, and hepatocellular carcinoma (HCC).

Personalized Assessment of Mortality Risk and Hospital Stay Duration in Hospitalized Patients with COVID-19 Treated with Remdesivir: A Machine Learning Approach.

Background: Despite advancements in vaccination, early treatments, and understanding of SARS-CoV-2, its impact remains significant worldwide. Many patients require intensive care due to severe COVID-19. Remdesivir, a key treatment option among viral RNA polymerase inhibitors, lacks comprehensive studies on factors associated with its effectiveness. Methods: We conducted a retrospective study in 2022, analyzing data from 252 hospitalized COVID-19 patients treated with remdesivir. Six machine learning algorithms were compared to predict factors influencing remdesivir's clinical benefits regarding mortality and hospital stay. Results: The extreme gradient boost (XGB) method showed the highest accuracy for both mortality (95.45%) and hospital stay (94.24%). Factors associated with worse outcomes in terms of mortality included limitations in life support, ventilatory support needs, lymphopenia, low albumin and hemoglobin levels, flu and/or coinfection, and cough. For hospital stay, factors included vaccine doses, lung density, pulmonary radiological status, comorbidities, oxygen therapy, troponin, lactate dehydrogenase levels, and asthenia. Conclusions: These findings underscore XGB's effectiveness in accurately categorizing COVID-19 patients undergoing remdesivir treatment.

Treatment with Remdesivir of Children with SARS-CoV-2 Infection: Experience from a Clinical Hospital in Romania.

The COVID-19 pandemic was characterized by mild-to-moderate disease in children and adolescents, with low incidences of severe cases and mortality. Most of the information on drug therapy in COVID-19-positive children was derived from research in adult patients. Remdesivir, an inhibitor of viral RNA polymerase, was shown to be effective in COVID-19 patients with moderate-to-severe disease. In this study, we present our experience of the use of remdesivir in pediatric patients hospitalized with COVID-19. This retrospective study was based on the early use of remdesivir in 14 children with mild, moderate, and severe clinical forms of COVID-19, who were hospitalized between 1 January 2022, and 30 September 2023. The patients included eight infants and six children older than 1 day (the age range was 2 months to 17 years). Most of them (92.85%) had documented pneumonia. Four patients had associated acute laryngitis, and another had bronchiolitis. Coinfections with Streptococcus pneumoniae were diagnosed in two patients. The clinical course was favorable in 12/14 (85.71%) children. Two patients were transferred to the pediatric intensive care unit because of aggravation of associated acute diseases (acute laryngitis and bronchiolitis, respectively). Mild increases in alanine aminotransferase levels occurred in two patients, with no increase in serum creatinine, during treatment with remdesivir. The appropriate use of remdesivir proved safe and efficient in our group of patients. However, further studies are required to support the efficiency, tolerability, and safety of remdesivir in children.

Активні компоненти флавоноїдного складу «Протефлазид» гальмують репродукцію вірусу грипу: можливі молекулярні мішені взаємодії

The search for substances possessing antiviral activities, in particular anti-influenza activity, is of importance for designing new drugs that may be effective in combating viral infections. The antiviral substances of the natural origin such as flavonoids and their derivatives are in the focus of numerous studies. The topical problem is the elucidation of the mechanisms of the interaction between flavonoid substances and the virus-specific targets in infected cells. Aim. To assess the activity of the flavonoid-enriched composition containing the biologically active substances of Proteflazidum against influenza virus in vitro and in vivo and to analyze in silico the putative interactions of the flavonoid components of the composition with PB2 subunit of viral RNA-polymerase. Methods. The anti-influenza effects of flavonoid-enriched composition prepared from the extracts of Deschampsia caespitosa L. and Calamagrostis epigeios L. were assessed in vitro in MDCK cells and in vivo in white outbred mice. Virion RNA was analyzed by RT-PCR with the primers detecting the transcripts of PB1 and PB2 subunits of viral RNA-polymerase and hemagglutinin. The potential interaction of the representative flavonoids of the composition with PB2 subunit of RNA-polymerase was analyzed in silico by molecular docking. Results. The composition under study inhibits effectively replication of А/FM/1/47 (H1N1) strain of influenza virus in vitro and protects the mice against flu infection both in therapeutic and preventive modes of its administration. According to the molecular docking findings, all three major flavonoid compounds of the composition, quercetin, luteolin, and apigenin, interact similarly with the PB2 domain of viral RNA-polymerase. Conclusions. The flavonoid composition containing the biologically active substances of Proteflazidum could be considered as the anti-flu drug with the PB2 subunit of viral RNA-polymerase being one of its potential molecular targets.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

An alpha-herpesvirus employs host HEXIM1 to promote viral transcription.

Although it is widely accepted that herpesviruses utilize host RNA polymerase II (RNAPII) to transcribe viral genes, the mechanism of utilization varies significantly among herpesviruses. With the exception of herpes simplex virus 1 (HSV-1) in alpha-herpesviruses, the mechanism by which RNAPII transcribes viral genes in the remaining alpha-herpesviruses has not been reported. In this study, we investigated the transcriptional mechanism of an avian alpha-herpesvirus, Anatid herpesvirus 1 (AnHV-1). We discovered for the first time that hexamethylene-bis-acetamide-inducing protein 1 (HEXIM1), a major inhibitor of positive elongation factor B (P-TEFb), was significantly upregulated during AnHV-1 infection, and its expression was dynamically regulated throughout the progression of the disease. However, the expression level of HEXIM1 remained stable before and after HSV-1 infection. Excessive HEXIM1 assists AnHV-1 in progeny virus production, gene expression, and RNA polymerase II recruitment by promoting the formation of more inactive P-TEFb and the loss of RNAPII S2 phosphorylation. Conversely, the expression of some host survival-related genes, such as SOX8, CDK1, MYC, and ID2, was suppressed by HEXIM1 overexpression. Further investigation revealed that the C-terminus of the AnHV-1 US1 gene is responsible for the upregulation of HEXIM1 by activating its promoter but not by interacting with P-TEFb, which is the mechanism adopted by its homologs, HSV-1 ICP22. Additionally, the virus proliferation deficiency caused by US1 deletion during the early infection stage could be partially rescued by HEXIM1 overexpression, suggesting that HEXIM1 is responsible for AnHV-1 gaining transcription advantages when competing with cells. Taken together, this study revealed a novel HEXIM1-dependent AnHV-1 transcription mechanism, which has not been previously reported in herpesvirus or even DNA virus studies.IMPORTANCEHexamethylene-bis-acetamide-inducing protein 1 (HEXIM1) has been identified as an inhibitor of positive transcriptional elongation factor b associated with cancer, AIDS, myocardial hypertrophy, and inflammation. Surprisingly, no previous reports have explored the role of HEXIM1 in herpesvirus transcription. This study reveals a mechanism distinct from the currently known herpesvirus utilization of RNA polymerase II, highlighting the dependence on high HEXIM1 expression, which may be a previously unrecognized facet of the host shutoff manifested by many DNA viruses. Moreover, this discovery expands the significance of HEXIM1 in pathogen infection. It raises intriguing questions about whether other herpesviruses employ similar mechanisms to manipulate HEXIM1 and if this molecular target can be exploited to limit productive replication. Thus, this discovery not only contributes to our understanding of herpesvirus infection regulation but also holds implications for broader research on other herpesviruses, even DNA viruses.

Genistein is effective in inhibiting Orf virus infection in vitro by targeting viral RNA polymerase subunit RPO30 protein.

Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.

Avian ANP32A incorporated in avian influenza A virions promotes interspecies transmission by priming early viral replication in mammals.

Species-specific differences in acidic nuclear phosphoprotein 32 family member A (ANP32A) determine the restriction of avian-signature polymerase in mammalian cells. Mutations that evade this restriction, such as PB2-E627K, are frequently acquired when avian influenza A viruses jump from avian hosts to mammalian hosts. However, the mechanism underlying this adaptation process is still unclear. Here, we report that host factor ANP32 proteins can be incorporated into influenza viral particles through combination with the viral RNA polymerase (vPol) and then transferred into targeted cells where they support virus replication. The packaging of the ANP32 proteins into influenza viruses is dependent on their affinity with the vPol. Avian ANP32A (avANP32A) delivered by avian influenza A virions primes early viral replication in mammalian cells, thereby favoring the downstream interspecies transmission event by increasing the total amount of virus carrying adaptive mutations. Our study clarifies one role of avANP32A where it is used by avian influenza virus to help counteract the restriction barrier in mammals.

The interaction of influenza A virus RNA polymerase PA subunit with the human β-actin protein

Influenza A viruses are enveloped viruses with a genome of eight single-stranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxy-terminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription.

In silico analysis of identified molecules using LC–HR/MS of Cupressus sempervirens L. ethyl acetate fraction against three monkeypox virus targets

Monkeypox virus is a viral disease transmitted to humans through contact with infected animals, such as monkeys and rodents, or through direct contact with the bodily fluids or lesions of infected humans. The aim of this study is to evaluate in silico the inhibition effect of eight Cupressus sempervirens L. ethyl acetate fraction identified molecules using LC-MS on three monkeypox targets such as the vaccinia virus thymidylate kinase (VTK), the viral profilin-like protein (VPP), and the viral RNA polymerase (VRP). The study consist of using molecular docking with AutoDock vina based on the lowest energy value in kcal/mol, pharmacokinetics prediction with pre-ADMET v2.0 server, and prediction of biological activity with the PASS server tool. The best complexes were subjected to molecular dynamics simulation (MD) study to confirm their stability using Desmond software. The used molecules were vitamin C, vanillic acid (Pol), Flav1 (Catechin), Flav2 (Epicatechin), Flav3 (Hyperoside), Flav4 (Luteolin), Flav5 (Taxifolin), and Flav6 (Quercetin). The results show that flavonoids are potent to VTK, VPP and effectively block the VRP channel with energy values ranging from −7.0 to −9.3 kcal/mol. Further, MD simulation supports Flav1 and, Flav2 for notable stability in the VTK binding pocket through hydrogen and hydrophobic interactions. PASS results predicted various biological activities with promising VTK and VRP inhibition activities. The studied molecules could constitute a safer alternative to current drugs, which often cause adverse side effects. Communicated by Ramaswamy H. Sarma

Defining the minimal components of the influenza A virus replication machinery via an in vitro reconstitution system.

During influenza A virus infection, the viral RNA polymerase transcribes the viral negative-sense segmented RNA genome and replicates it in a two-step process via complementary RNA within viral ribonucleoprotein (vRNP) complexes. While numerous viral and host factors involved in vRNP functions have been identified, dissecting the roles of individual factors remains challenging due to the complex cellular environment in which vRNP activity has been studied. To overcome this challenge, we reconstituted viral transcription and a full cycle of replication in a test tube using vRNPs isolated from virions and recombinant factors essential for these processes. This novel system uncovers the minimal components required for influenza virus replication and also reveals new roles of regulatory factors in viral replication. Moreover, it sheds light on the molecular interplay underlying the temporal regulation of viral transcription and replication. Our highly robust in vitro system enables systematic functional analysis of factors modulating influenza virus vRNP activity and paves the way for imaging key steps of viral transcription and replication.

Equine ANP32 proteins support influenza A virus RNA polymerase activity

Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses. To date, the molecular properties of equine ANP32 (eqANP32) protein are poorly understood, particularly the mechanisms that affect equine influenza virus (EIV) RNA polymerase activity. Here, we found that there are six alternative splicing variants of equine ANP32A (eqANP32A) with different levels of expression. Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees, with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity. Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation, but did not affect their ability to bind RNA polymerase. We also demonstrated that there is only one transcript of eqANP32B, and that this transcript showed only very low support to the EIV RNA polymerase. This functional defect in eqANP32B is caused by the sequence of the 110–259 amino acids at its C-terminus. Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses. In conclusion, our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B, suggesting for the first time that the N-Cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.

Galectin 9 restricts viral replication in teleost via autophagy-antiviral pathway and polarizes M2 macrophages for anti-inflammatory response: New insights into functional properties of fish Galectin-9 from Planiliza haematocheilus

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 μg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Negative and ambisense RNA virus ribonucleocapsids: more than protective armor.

SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.

Transcriptional Stochasticity as a Key Aspect of HIV-1 Latency.

This review summarizes current advances in the role of transcriptional stochasticity in HIV-1 latency, which were possible in a large part due to the development of single-cell approaches. HIV-1 transcription proceeds in bursts of RNA production, which stem from the stochastic switching of the viral promoter between ON and OFF states. This switching is caused by random binding dynamics of transcription factors and nucleosomes to the viral promoter and occurs at several time scales from minutes to hours. Transcriptional bursts are mainly controlled by the core transcription factors TBP, SP1 and NF-κb, the chromatin status of the viral promoter and RNA polymerase II pausing. In particular, spontaneous variability in the promoter chromatin creates heterogeneity in the response to activators such as TNF-α, which is then amplified by the Tat feedback loop to generate high and low viral transcriptional states. This phenomenon is likely at the basis of the partial and stochastic response of latent T cells from HIV-1 patients to latency-reversing agents, which is a barrier for the development of shock-and-kill strategies of viral eradication. A detailed understanding of the transcriptional stochasticity of HIV-1 and the possibility to precisely model this phenomenon will be important assets to develop more effective therapeutic strategies.

Hardening of Respiratory Syncytial Virus Inclusion Bodies by Cyclopamine Proceeds through Perturbation of the Interactions of the M2-1 Protein with RNA and the P Protein.

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.

Construction and characterization of a new hepatitis C virus genotype 6a subgenomic replicon that is prone to render the sofosbuvir resistance.

Hepatitis C virus (HCV) infection remains a challenge to human public health despite the development of highly effective direct-acting antivirals (DAAs). Sofosbuvir (SOF), a key component in most DAA-based anti-HCV cocktail regimens, is a potent viral RNA polymerase (NS5B) inhibitor with a high barrier to drug resistance. The serine-to-threonine mutation at NS5B 282 (S282T) confers the SOF resistance, but severely impairs viral replication in most HCV genotypes (GTs) and cannot be stably maintained after the termination of the SOF-based therapies. In this study, we first developed a new HCV GT-6a subgenomic replicon PR58D6. Next, we selected SOF-resistant PR58D6 variants by culturing the replicon cells in the presence of SOF. Interestingly, unlike many other HCV replicons which require additional mutations to compensate for the S282T-inducing fitness loss, S282T alone in PR58D6 is genetically stable and confers the SOF resistance without significantly impairing viral replication. Furthermore, we showed that amino acid residue at NS5B 74 (R74) and 556 (D556) which are conserved in GT 6a HCV contribute to efficient replication of PR58D6 containing S282T. Finally, we showed that the G556D mutation in NS5B could rescue the replication deficiency of the S282T in JFH1, a GT-2a replicon. In conclusion, we showed that a novel GT-6a HCV replicon may easily render SOF resistance, which may call for attention to potential drug resistance during DAA therapies of HCV GT-6a patients.

Comparative analysis of PB2 residue 627E/K/V in H5 subtypes of avian influenza viruses isolated from birds and mammals.

Avian influenza viruses (AIVs) are naturally found in wild birds, primarily in migratory waterfowl. Although species barriers exist, many AIVs have demonstrated the ability to jump from bird species to mammalian species. A key contributor to this jump is the adaption of the viral RNA polymerase complex to a new host for efficient replication of its RNA genome. The AIV PB2 gene appears to be essential in this conversion, as key residues have been discovered at amino acid position 627 that interact with the host cellular protein, acidic nuclear phosphoprotein 32 family member A (ANP32A). In particular, the conversion of glutamic acid (E) to lysine (K) is frequently observed at this position following isolation in mammals. The focus of this report was to compare the distribution of PB2 627 residues from different lineages and origins of H5 AIV, determine the prevalence between historical and contemporary sequences, and investigate the ratio of amino acids in avian vs. mammalian AIV sequences. Results demonstrate a low prevalence of E627K in H5 non-Goose/Guangdong/1996-lineage (Gs/GD) AIV samples, with a low number of mammalian sequences in general. In contrast, the H5-Gs/GD lineage sequences had an increased prevalence of the E627K mutation and contained more mammalian sequences. An approximate 40% conversion of E to K was observed in human sequences of H5 AIV, suggesting a non-exclusive requirement. Taken together, these results expand our understanding of the distribution of these residues within different subtypes of AIV and aid in our knowledge of PB2 mutations in different species.

Evolution oftransient RNA structure-RNA polymerase interactions inrespiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.