Mechanism Of Viral Replication Research Articles

Virtual screening-driven drug discovery of SARS-CoV2 enzyme inhibitors targeting viral attachment, replication, post-translational modification and host immunity evasion infection mechanisms

The novel coronavirus SARS-CoV2, the causative agent of the pandemic disease COVID-19, emerged in December 2019 forcing lockdown of communities in many countries. The absence of specific drugs and vaccines, the rapid transmission of the virus, and the increasing number of deaths worldwide necessitated the discovery of new substances for anti-COVID-19 drug development. With the aid of bioinformatics and computational modelling, ninety seven antiviral secondary metabolites from fungi were docked onto five SARS-CoV2 enzymes involved in viral attachment, replication, post-translational modification, and host immunity evasion infection mechanisms followed by molecular dynamics simulation and in silico ADMET prediction (absorption, distribution, metabolism, excretion and toxicity) of the hit compounds. Thus, three fumiquinazoline alkaloids scedapin C (15), quinadoline B (19) and norquinadoline A (20), the polyketide isochaetochromin D1 (8), and the terpenoid 11a-dehydroxyisoterreulactone A (11) exhibited high binding affinities on the target proteins, papain-like protease (PLpro), chymotrypsin-like protease (3CLpro), RNA-directed RNA polymerase (RdRp), non-structural protein 15 (nsp15), and the spike binding domain to GRP78. Molecular dynamics simulation was performed to optimize the interaction and investigate the stability of the top-scoring ligands in complex with the five target proteins. All tested complexes were found to have dynamic stability. Of the five top-scoring metabolites, quinadoline B (19) was predicted to confer favorable ADMET values, high gastrointestinal absorptive probability and poor blood-brain barrier crossing capacities. Communicated by Ramaswamy H. Sarma

COVID-19, treatment in adults.

A finales de 2019, se reportaron casos de COVID-19, en la ciudad de Wuhan, China, un nuevo coronavirus, que se originó en animales como el murciélago, pangolín y el contacto humano con estos animales. La principal vía de transmisión entre seres humanos es la respiratoria, pero aún se estudian otros mecanismos. El SARS-CoV-2, es un betacoronavirus, cuyo genoma es ARN monocatenario, envuelto por proteínas M, E y S, esta última responsable de la unión a receptores de células humanas. Los tratamientos disponibles se basan en contrarrestar al virus (antivirales) y en potenciar los mecanismos de defensa del organismo (inmuno moduladores), así como bloquear los mecanismos de replicación viral en el huésped. La mayoría de estos tratamientos se encuentran en investigación y ninguno ha sido demostrado como efectivo y seguro. Se realiza un análisis de las opciones terapéuticas disponibles.

Raymond L. Erikson, PhD: In Memoriam (1936–2020)

![][1] > I hoed and trenched and weeded, And took the flowers to fair. > > The Shropshire Lad by A. E. Houseman In 2016, Ray started a personal reflection on his career in science titled “Four Decades of Protein Phosphorylation and Cancer Research.” Nothing more aptly fit his

Establishment of a Reverse Genetics System for Influenza D Virus.

Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

Enterovirus D68 Antivirals: Past, Present, and Future.

Enterovirus D68 (EV-D68) is an RNA virus that causes respiratory illnesses mainly in children. In severe cases, it can lead to neurological complications such as acute flaccid myelitis (AFM). EV-D68 belongs to the enterovirus genera of the Picornaviridae family, which also includes many other significant human pathogens such as poliovirus, enterovirus A71, and rhinovirus. There are currently no vaccines or antivirals against EV-D68. In this review, we present the current understanding of the link between EV-D68 and AFM, the mechanism of viral replication, and recent progress in developing EV-D68 antivirals by targeting various viral proteins and host factors that are essential for viral replication. The future directions of EV-D68 antiviral drug discovery and the criteria for drugs to reach clinical trials are also discussed.

Combinatorial Minigenome Systems for Emerging Banyangviruses Reveal Viral Reassortment Potential and Importance of a Protruding Nucleotide in Genome "Panhandle" for Promoter Activity and Reassortment.

Banyangvirus is a new genus (Phenuiviridae family, Bunyavirales order) that comprises a group of emerging tick-borne viruses with severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) as virulent representatives. As segmented RNA viruses, bunyaviruses may have genome reassortment potential, increasing the concern about new life-threatening bunyavirus emergence. Using a series of combinatory minigenome reporter assays based on transfection and superinfection, we showed that replication machinery proteins of designated banyangviruses can recognize genomic untranslated regions (UTRs) of other banyangviruses and assemble heterogenous minigenomes into functional ribonucleoproteins (RNPs). Moreover, both heterogenous and heterozygous RNPs were efficiently packaged by viral glycoproteins into infectious virus-like particles, manifesting remarkable reassortment potential of banyangviruses. Meanwhile, UTR promoter strength of the three banyangvirus segments appeared to be M > L > S. Secondary structure analysis revealed a conservative non-basepairing protruding nucleotide in the terminal UTR panhandles of M and L (but not S) segments of all banyangviruses and some related phleboviruses (Phlebovirus genus). Furthermore, not only a conserved panhandle region but also the protruding nucleotide proved important for UTR function. Removal of the protruding nucleotide abated M and L UTR activities and compatibilities with heterogenous viral proteins, and introduction of a protruding nucleotide into S panhandle, conversely, enhanced UTR promoter strength and compatibility, revealing the significance of the protruding nucleotide as a new signature of the genomic panhandle structure in both UTR activity and reassortment potential. The study demonstrates not only banyangvirus reassortment potential but also the notable role of the protruding nucleotide in UTR function and reassortment, providing clues to viral evolution and replication mechanisms and perhaps benefiting disease control and prevention in the future.

HIV‐1 Matrix Protein Interactions with Cellular tRNAs

The human immunodeficiency virus (HIV) is a retrovirus that infects roughly 40 million people globally that attacks CD4+ T‐cells, thus impairing the immune system. Highly active antiretroviral treatment against the virus is available; however, the viral genome frequently mutates, which gives rise to evasion of current therapies and highlights the need for new drugs. Better understanding molecular mechanisms of viral replication may lend to this effort. One process necessary for HIV replication is membrane trafficking of the Gag polyprotein (Gag) to the plasma membrane via Gag’s N‐terminal matrix domain (MA). MA mediates membrane targeting by means of electrostatic interaction and anchoring to the plasma membrane using a covalently linked myristoyl moiety. Recent virology studies demonstrated that MA interacts with cellular tRNAs, including tRNALys3 and tRNAGlyGCC, before binding to the plasma membrane, but the role of tRNA on assembly is poorly understood. The long‐term goal of this work is to study the MA:tRNA complex and elucidate its 3D structure as characterization of this interaction may shed light on the function of tRNA during membrane targeting. To achieve this goal, conditions to stabilize MA and the tRNA independently and in complex must be established. Nuclear magnetic resonance (NMR) and gel electrophoresis were employed to identify conditions that stabilized the tRNA and MA independently. Electrophoretic mobility shift assays (EMSAs) were carried out to probe MA:tRNA interactions under these conditions and showed complex formation upon addition of a stoichiometric ratio of MA to tRNA. This work lays a foundation to study thermodynamics of the MA‐tRNA interaction and determine the three‐dimensional structure of the complex.Support or Funding InformationThis research was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC, HHMI, and NIH grants R01GM042561 and 8R01AI50498.

In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy

Rationale: Zika virus (ZIKV) is a pathogenic virus known to cause a wide range of congenital abnormalities, including microcephaly, Guillain-Barre syndrome, meningoencephalitis, and other neurological complications, in humans. This study investigated the noninvasive detection of ZIKV infection in vivo, which is necessary for elucidating the virus's mechanisms of viral replication and pathogenesis, as well as to accelerate the development of anti-ZIKV therapeutic strategies.Methods: In this study, a recombinant ZIKV harbouring Nluc gene (ZIKV-Nluc) was designed, recovered, and purified. The levels of bioluminescence were directly correlated with viral loads in vitro and in vivo. The dynamics of ZIKV infection in A129 (interferon (IFN)-α/β receptor deficient), AG6 (IFN-α/β and IFN-γ receptor deficient), and C57BL/6 mice were characterized. Pregnant dams were infected with ZIKV-Nluc at E10 via intra footpad injection. Then, the pooled immune sera (anti-ZIKV neutralizing antibodies) #22-1 in ZIKV-Nluc virus-infected mice were visualized.Results: ZIKV-Nluc showed a high genetic stability and replicated well in cells with similar properties to the wild-type ZIKV (ZIKVwt). Striking bioluminescence signals were consistently observed in animal organs, including spleen, intestine, testis, uterus/ovary, and kidney. The ileocecal junction was found to be the crucial visceral target. Infection of pregnant dams with ZIKV-Nluc showed that ZIKV was capable of crossing the maternal-fetal barrier to infect the fetuses via vertical transmission. Furthermore, it was visualized that treatment with the pooled immune sera was found to greatly restrict the spread of the ZIKV-Nluc virus in mice.Conclusions: This study is the first to report the real-time noninvasive tracking of the progression of ZIKV invading immune-sheltered tissues and propagating vertically during pregnancy. The results demonstrate that ZIKV-Nluc represents a powerful tool for the study of the replication, dissemination, pathogenesis, and treatment of ZIKV in vitro and in vivo.

The Effect of the Infection Rate on Oncolytic Virotherapy

Oncolytic viruses have become a novel therapeutic tool for various cancer treatments. Several naturally occurring oncolytic viruses and engineered oncolytic viruses are developed for oncolytic virotherapies. Although we have a good understanding on molecular mechanisms of viral replication and virus-induced cell lysis at the cellular level, it is unclear how oncolytic viruses and cancer cells interact as a population. Several mathematical models of oncolytic virotherapy have been developed to advance the understanding of dynamic interaction between oncolytic viruses and cancer cells. Many authors investigated the effect of the virus replication on dynamics of cancer cell population and proposed that the bursting rate of viruses is an important factor for successful oncolytic virotherapy. In this study, we investigate the effect of infection rate of oncolytic viruses on an oncolytic virotherapy model. Particularly, we focused on studying the relationship between two control parameters, bursting rate and infection rate of the virus, to generate the patterns from equilibrium steady state to periodic solutions. Based on the model, the interaction between cancer cells and oncolytic viruses shows an intriguing two-dimensional bifurcation, showing three parameter regions (equilibrium steady state, damped oscillations and oscillations). Our result suggests that both infection rate and bursting rate are crucial properties of oncolytic viruses to design a successful oncolytic virotherapy.

Structure and Function of the Influenza Virus Transcription and Replication Machinery.

Transcription and replication of the influenza virus RNA genome is catalyzed by the viral heterotrimeric RNA-dependent RNA polymerase in the context of viral ribonucleoprotein (vRNP) complexes. Atomic resolution structures of the viral RNA synthesis machinery have offered insights into the initiation mechanisms of viral transcription and genome replication, and the interaction of the viral RNA polymerase with host RNA polymerase II, which is required for the initiation of viral transcription. Replication of the viral RNA genome by the viral RNA polymerase depends on host ANP32A, and host-specific sequence differences in ANP32A underlie the poor activity of avian influenza virus polymerases in mammalian cells. A failure to faithfully copy the viral genome segments can lead to the production of aberrant viral RNA products, such as defective interfering (DI) RNAs and mini viral RNAs (mvRNAs). Both aberrant RNA types have been implicated in innate immune responses against influenza virus infection. This review discusses recent insights into the structure-function relationship of the viral RNA polymerase and its role in determining host range and virulence.

Identification of candidate molecular determinants of the vector competence of Ixodes ricinus for members of the tick-borne encephalitis complex

In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells. To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod. This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons.

Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-κB signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species.IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

Background. The specific detection of genomic/template +RNA and replicative –RNA of tick-born encephalitis virus (TBEV) is necessary to study the mechanisms of viral replication in the cells of reservoir and accidental hosts. However, the current approaches of quantitative reverse transcription – polymerase chain reaction (qRT-PCR) are rather focused on the detection of total viral RNA load in the sample. Thus, the significant optimization is necessary both for RT-PCR and for RNA copy number standard preparation.Aims. To develop the set of standard samples of synthetic +RNA of TBEV and to optimize qRT-PCR for quantification of genomic +RNA of the virus.Materials and methods. Fragment of the genomic +RNA of TBEV was synthesized using pTZ57R-T\A plasmid vector with embedded T7 promoter and T7 RNA polymerase. The DNA contamination was removed using RNase-free DNase I treatment followed by additional RNA purification step. Reverse transcription was performed using specific antisense primer 11154R 5`- AGCGGGTGTTTTTCCG-3` and qPCR detection was used according to the modified procedure of M. Schwaiger and P. Cassinotti (2003).Results. As a result of the amplification of standard samples, the concentration of positive polarity ТBEV RNA, carried out in five independent repetitions on different days, the correlation coefficient R2 between the quantification cycle and the concentration of the standard sample was 0.99, and the efficiency of PCR was 100 %. The coefficient of variation in assessing the inter-test accuracy of determination averaged 2.8 %.Conclusions. Optimized qRT-PCR procedure and set of +RNA standards allow to determine the concentration of genomic +RNA of TBEV in routine laboratory practice.

Development of antiviral drugs against hepatitis B virus

Hepatitis B virus (HBV) infection is prevalent throughout the world, with approximately 300 million people chronically infected. HBV infection can cause acute and chronic liver hepatitis. Long-term chronic infection can increase the incidence of liver cirrhosis and hepatocellular carcinoma (HCC). At present, there are two types of approved drugs for chronic HBV infection: nucleos(t)ide analogs (NAs) and interferon α (IFNα), currently pegylated IFNα (PegIFNα). Although these drugs can inhibit viral replication and reduce the risk of liver cirrhosis and HCC, the limitations are obvious: HBV surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) pool cannot be efficiently eliminated. To overcome these limitations, new drugs are required urgently to be designed and developed. In recent years, with the deepening of research on HBV, drugs targeting different targets of viral complex replication mechanisms and interactions with the host immune system have been developed. Drugs targeting viral complex replication mechanisms include HBV entry inhibitors, cccDNA formation and transcription inhibitors, gene editing targeting HBV DNA, HBV specific siRNAs, capsid inhibitors, polymerase inhibitors and HBV secretion inhibitors. Entry inhibitors have been used successfully in treating viral infections. Myrcludex B, a synthetic N-myristoylated lipopeptide derived from HBV preS1 protein, competes with the virus for binding to the solute carrier family 10 member 1 (SLC10A1 or NTCP). This agent prevents HBV and Hepatitis D virus (HDV) infection and in cells and animal models. cccDNA is the transcriptional template of HBV RNAs and is an ideal target for clearing HBV. DNA ligase inhibitors, which can inhibit cccDNA formation and are being developed as anticancer agents, may be tested for treatment of chronic HBV infection, if the side effects are tolerable. The genome editing tool, clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9), has many innovative applications in different fields because of its potential for gene therapy. These have provided a clear proof of concept that this approach has the potential to treat or even cure patients with chronic HBV infection. RNA interference technology can be used to manipulate virus gene expression and may be developed for HBV treatment. HBV nucleocapsid formation and pgRNA packaging are critical steps of viral life cycle that may be targeted by antiviral agents. Inside the viral capsid, pgRNA is reverse transcribed into minus-strand DNA by the viral polymerase, with its terminal protein domain acting as a primer for initiation of reverse transcription. HBV polymerase is also a good drug target. Drugs targeting the host immune system include that stimulating the innate immune system, like IFNα, Toll-like receptor (TLR) agonists and stimulating cytoplasmic nucleic acid sensors, and that activating an adaptive immune response, like checkpoint inhibitors, T-cell redirection and therapeutic vaccines. IFN-based therapies are considered as immunomodulatory strategies. TLRs are expressed either at the cell surface or on endosome membranes, where they recognize molecular patterns associated with invading pathogens. Stimulation of TLR1, TLR2, TLR3, and TLR4 on hepatocytes has direct antiviral effects against HBV. The RNA helicases DExD/H-box helicase 58 (DDX58, also called RIG-I) and IFN induced with helicase C domain 1 (IFIH1, also called MDA5) are RNA sensors in the cytoplasm that induce production of cytokines by activating IRF3 and NF-kB. Activation of RIG-I induces an antiviral response against HBV. PD-1 is the inhibitory receptor expressed by most HBV specific T cells, so agents are in development to disrupt the interaction between PD-1 and its ligand, PD-L1. A therapeutic vaccine against HBV needs to overcome mechanisms of HBV-specific immune tolerance. Because natural resolution of HBV infection is accompanied by lifelong T-cell- and antibody-mediated immunity, these vaccines should induce HBV-specific B- and T-cell immune responses. Here, we review recent advances in development of directing-acting antiviral (DAA), host-targeting agents and immunomodulatory therapies. Some of which have entered clinical trials. We also discuss strategies for unbiased high-throughput screens to identify compounds that inhibit HBV and for repurposing existing drugs.

Implication of Viral Infections for Greenhouse Gas Dynamics in Freshwater Wetlands: Challenges and Perspectives.

Viruses are non-living, acellular entities, and the most abundant biological agents on earth. They are widely acknowledged as having the capacity to influence global biogeochemical cycles by infecting the bacterial and archaeal populations that regulate carbon and nutrient turnover. Evidence suggests that the majority of viruses in wetlands are bacteriophages, but despite their importance, studies on how viruses control the prokaryotic community and the concomitant impacts on ecosystem function (such as carbon cycling and greenhouse gas flux) in wetlands are rare. Here we investigate virus-prokaryote interactions in freshwater wetland ecosystems in the context of their potential influence on biogeochemical cycling. Specifically, we (1) synthesize existing literature to establish current understanding of virus-prokaryote interactions, focusing on the implications for wetland greenhouse gas dynamics and (2) identify future research priorities. Viral dynamics in freshwater wetlands have received much less attention compared to those in marine ecosystems. However, based on our literature review, within the last 10 years, viral ecology studies on freshwater wetlands have increased twofold. Despite this increase in literature, the potential implication of viral infections on greenhouse gas emission dynamics is still a knowledge gap. We hypothesize that the rate of greenhouse gas emissions and the pool of sequestered carbon could be strongly linked to the type and rate of viral infection. Viral replication mechanism choice will consequently influence the microbial efficiency of organic matter assimilation and thus the ultimate fate of carbon as a greenhouse gas or stored in soils.

Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein’s broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

Advances in Tetravirus Research: New Insight Into the Infectious Virus Lifecycle and an Expanding Host Range.

Tetraviruses are a group of relatively unknown small RNA viruses with particles that display a characteristic T=4 capsid architecture. Tetraviruses are classified into three families, the Alphatetraviridae, Permutotetraviridae and Carmotetraviridae, according to the divergent characteristics of their respective viral replicases. Tetraviruses generally infect the larvae of lepidopteran insect species, many of which are important agricultural pests and, until recently, were thought to have an unusually narrow host range and tissue tropism. The development of experimental systems for studying the viral infectious life cycle in tissue culture has permitted the extension of the virus host range to mammalian cells and plants. This chapter will review recent advances in the understanding of the biology of tetraviruses, highlighting new information on the expression and functional characterisation of viral proteins and the development of biological systems for elucidating the molecular mechanisms of infection, viral replication and host range.

Community-acquired respiratory viruses.

Incidence of community-acquired respiratory viruses (CARVs) after lung transplantation (LTx) is 10-68 cases per 100 patient-years. Infected patients may develop graft failure and there seems to be an association between CARV infection and development of chronic lung allograft dysfunction (CLAD). This review summarizes the impact of CARV infection after LTx and potential treatment strategies. Detection rate of CARV depends on diagnostic methods. CARV infections after LTx are reported more frequently probably attributed to improved diagnostic methods, especially nucleic acid testing. Paramyxoviridae and picornaviridae are most frequent. For paramyxoviridae, the association with CLAD is reported in various single-center observational studies. Neuraminidase inhibitors are approved for influenza and can be safely used in flu-infected LTx patients. There is no approved treatment for paramyxoviruses, most centers use ribavirin in the infected LTx recipient. Antivirals against CARV in LTx recipients have not yet demonstrated reduced morbidity in randomized clinical trials. Agents against CARV under development are inhibiting viral attachment and use silencing mechanisms of viral replication. The cohort of lung transplant recipients is a focus of intense research because of the high morbidity of CARV infection and intense surveillance of LTx recipeints.

Dimerisation of the influenza virus RNA polymerase during viral genome replication

Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.

Phosphorylation of nucleopolyhedrovirus 39K is essential for the regulation of viral gene transcription in silkworm cells.

39K protein was proven to be the most notable hyperphosphorylated protein of Bombyx mori nucleopolyhedrovirus (BmNPV), since its amino acid 136 has the highest phosphorylation ratio (16.683) among all of BmNPV phosphorylated proteins (Shobahah et al., 2017). Therefore, we inserted positive (mimicking phosphorylation) and negative (mimicking dephosphorylation) mutants of the highest phosphorylated site of 39K and the wild type 39k into the 39k-ko-Bacmid using the Bac-to-Bac system. These three kinds of recombinant Bacmids along with wild type and knocked-out Bacmid were then transfected into the Bombyx mori cells (BmN) and further investigated by qPCR analysis. The results of the qPCR showed that 39K phosphorylation had no significant effect on viral genome replication, unlike the positive mutation that reduced the viral gene transcription drastically compared to the wild type. Thus, phosphorylation of BmNPV 39K protein does not appear to be the essential mechanism for viral genome replication, even though it has an important role in the viral gene transcription. Keywords: BmNPV; 39K; phosphorylation; virus replication; virus transcription.