Viral Synthesis Research Articles

Molecular basis of quercetin as a plausible common denominator of macrophage-cholesterol-fenofibrate dependent potential COVID-19 treatment axis

The world's largest randomized control trial against COVID-19 using remdesivir, hydroxychloroquine, lopinavir and interferon-β1a appeared to have little or no effect on hospitalized COVID-19 patients. This has again led to search for alternate re-purposed drugs and/or effective “add-on” nutritional supplementation, which can complement or enhance the therapeutic effect of re-purposed drug. Focus has been shifted to therapeutic targets of severe acute respiratory syndrome coronavirus (SARS-CoV-2), which includes specific enzymes and regulators of lipid metabolism. Very recently, fenofibrate (cholesterol-lowering drug), suppressed the SARS-CoV-2 replication and pathogenesis by affecting the pathways of lipid metabolism in lung cells of COVID-19 patients. A preclinical study has shown synergistic effect of quercetin (a flavonoid) and fenofibrate in reducing the cholesterol content, which might be useful in COVID-19 treatment. Based on the scientific literature, use of quercetin and fenofibrate in COVID-19 seems meaningful in pharmaceutical and biomedical research, and warrants basic, experimental and clinical studies. In this article, we have summarized the contemporary findings about drug fenofibrate and its effect on membrane synthesis of COVID-19 virus along with emphasizing on possible synergistic effects of quercetin with fenofibrate.

Discovery of Drug Candidate from Various Natural Products as Potential Novel Dengue Virus Nonstructural Protein 5 (NS5) Inhibitor

Dengue is a severe health problem, especially in under-developing countries like Indonesia. The government and the community have made various efforts. However, the current programs are focusing on controlling disease vector. Medical treatment so far only treats the symptoms. On the other hand, the development of antiviral compounds has been dominated by synthetic compounds which may have serious side effects. The potential of natural compounds in Asia is still largely unexplored. Dengue virus nonstructural protein 5 (NS5) is highly conserved in all serotypes. This study aims to screen several potential natural compounds to be developed as specific antiviral compounds targeting DENV NS5. This research conducted by using in silico approaches including data mining, molecular docking, and 2D-3D visualization. About 170 compounds from 88 organisms analyzed to get the top nine compounds with the highest affinity against NS5 protein. The top nine compounds are from the halichondramide, trigocherriolide, quinadoline, tryptoquivaline, and zingiberene groups. The docking results showed that these potential compounds have an affinity of about 67-100% higher than ribavirin. The visualization results show that these compounds are grouped into three different binding sites. They are RdRP, MTase, and the junction. Compounds that bind in RdRP are thought to interfere with the process of RNA viral synthesis, compounds that bind to MTase are thought to disrupt the viral RNA capping process, while compounds that bind between the two domains are thought to interfere NS5 structural integrity. The compound with the highest affinity is halishigamide A. Halishigamide A is known to have antimicrobial properties, but the action mechanism is still unknown. Compared to the control drug, the halishigamide A tends to have more dynamic movement and interaction to the target protein’s binding site due to its residues’ chemicals motive. Together, these results showed the potency of natural products as specific antiviral compounds targeting DENV NS5.

The role of enzymatic activities of antiviral proteins from plants for action against plant pathogens.

Antiviral proteins (AVPs) from plants possess multiple activities, such as N-glycosidase, RNase, DNase enzymatic activity, and induce pathogenesis-related proteins, salicylic acid, superoxide dismutase, peroxidase, and catalase. The N-glycosidase activity releases the adenine residues from sarcin/ricin (S/R) loop of large subunit of ribosomes and interfere the host protein synthesis process and this activity has been attributed for antiviral activity in plant. It has been shown that AVP binds directly to viral genome-linked protein of plant viruses and interfere with protein synthesis of virus. AVPs also possess the RNase and DNase like activity and may be targeting nucleic acid of viruses directly. Recently, the antifungal, antibacterial, and antiinsect properties of AVPs have also been demonstrated. Gene encoding for AVPs has been used for the development of transgenic resistant crops to a broad range of plant pathogens and insect pests. However, the cytotoxicity has been observed in transgenic crops using AVP gene in some cases which can be a limiting factor for its application in agriculture. In this review, we have reviewed various aspects of AVPs particularly their characteristics, possible mode of action and application.

Effect of thermal helium-oxygen mixture on viral load in COVID-19

In this paper, we will discuss the use of t-Не/О 2 in the treatment of patients with the viral disease COVID-19. The aim of the study is to evaluate the effect of thermal helium-oxygen therapy on viral load, inflammatory markers, and antibody synthesis. Methods . A single-center, randomized, prospective study included 60 patients with COVID-19. Patients were divided into two groups: 1 ( n = 30; 17 male, 13 female) – t-Не/О 2 therapy was included in the standard COVID-19 treatment Protocol; 2 ( n = 30; 16 male, 14 female) – standard therapy in accordance with the clinical recommendations of Healthcare Ministry of Russia for patients with COVID-19. Of the 60 patients included in the study, 28 (46.7%) were medical professionals. The median age of patients in the study was 56.7 (45 – 61) years old. In the group 1 – 58 (45 – 59.5) years old, in the group 2 – 55 (46 – 66) years old. All patients had a positive test of SARS-CoV-2 coronavirus RNA, CT signs of “ground-glass opacity” type lung damage, and areas of air space consolidation. Patients were comparable by gender, age, body mass index (BMI), area of lesion of the pulmonary parenchyma, laboratory data. Results . As a result of the use of t-Не/О 2 , the elimination of the SARS-CoV-2 virus occurred within 48 – 72 hours from the start of inhalation and was confirmed by PCR test. The following changes were found in all patients: synthesis of IgM and IgG antibodies, increase in lymphocytes level, decrease of C-reactive protein, restoration of alanine aminotransferase and aspartate aminotransferase levels, D-dimer, and ferritin. These signs became more pronounced in the 1 st group within 72 – 168 hours, compared with the 2 nd group, where these results were achieved on the 10 th day of therapy. Conclusion . Тhe inclusion of thermal inhalation a gas mixture of helium and oxygen (t-Не/О 2 ) in the standard therapy of patients carrying infectious disease caused by SARS-CоV-2 with CT signs of COVID-19 pneumonia (СT1, CT2 grades) reduces the viral load by stimulating antibody synthesis, as the type of immunoglobulin G, and immunoglobulin M causing the effect of “termovaccination”; increases the effectiveness of treatment, reducing the markers of inflammation.

Role of structural and functional proteins of SARS -COV-2

Severe acute respiratory corona virus-2 (SARS-CoV-2) is a ribonucleic acid (RNA) virus with enveloped no-segmented positive sense belonging to a beta (β) - corona virus family. It has 29,903 nucleotides sized genome with 10 open reading frames (ORF). ORF1 (ab) encodes two polypeptides pp1a and pp1b cleaved into 16 functional proteins, which are mainly intended to form replication transcription complex (RTC). The cleavage process of pp1a and pp1b polypeptides to 16 functional proteins of SARs-CoV-2 is mainly facilitated by main protease and papain-like protease. The replication transcription complex (RTC) formed by the action of 16-functional proteins of SARs-CoV-2 is mainly involved as viral RNA synthesis machinery in the transcriptional and replication process of viral RNAs. ORF (2-10) encodes for structural (for example: spike (S), membrane (M), nucleocapsid (N), and envelop (E)) and accessory proteins of SARs-CoV-2. The main functions of structural proteins are viral assembly, viral coating, viral entry into host cells and assembly of the RNA genome. Accessory proteins are proteins that are not involved in the viral synthesis machinery, as 16 functional proteins, and in the viral assembly, coating, entry into host cells and packaging of Viral RNAs, as structural proteins. Rather, these are proteins that may play central role by enhancing viral assembly process, virulence and pathogenesis of SARs-CoV-2. Our aim in the current review was to elaborate the specific role of these structural and functional proteins on viral genomic replication and transcription, viral assembly, host cell attachment and pathogenesis. Multiple literatures have been reviewed to achieve the objective of this review.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex.

RNA-dependent RNA polymerases (RdRPs) from nonsegmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication, and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity, and in truncated L protein constructs, RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of an RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe nonradiometric assays for measuring RNA binding and RNA polymerization activity of RSV RdRP, which are amenable to compound screening and profiling.

Genotyping of Canine Distemper Virus Lineage in Clinically Infected Dogs in Puducherry, Southern India

Aims: This study aimed to determine the Canine Distemper Virus (CDV) lineage circulating in the Puducherry region (Southern India) and how they are genetically and antigenically related to the vaccine and other known CDV lineages around the world.
 Study Design: The study involved genetic characterization of the canine distemper virus strains/lineages circulating in the clinically suspected dogs in the field.
 Place and Duration of Study: Department of Veterinary Clinical Complex, Animal Husbandry Department, Private Veterinary Clinics, NGOs like Bark India, People for Animals organization, Puducherry between January 2018 and December 2019.
 Methodology: The ocular and nasal swabs were collected from 40 dogs suspected for canine distemper from Puducherry state (Southern India). Following viral RNA Extraction and cDNA synthesis; the cDNAs were screened for virus by targeting the CDV Nucleocapsid (N) gene using Reverse Transcriptase PCR. Further, the N gene positive cDNAs were genetically characterized for sequence analysis of the CDV hemagglutinin (H) gene.
 Results: A total of 15 (37.5%) out of 40 ocular/ nasal swabs were found to be CDV positive by RT-PCR targeting the N gene. The sequence analysis of the H gene revealed forty-nine non-synonymous and thirty-three synonymous mutations (out of 356 amino acids) in comparison with vaccine strain. The vaccine virus (Onderstepoort strain), which is related to the America 1 lineage, possessed high level of (30 – 35 i.e. more than 10%) amino acid divergence with the CDV sequences analysed in this study. The Phylogenetic analysis revealed that the present CDV sequences formed a separate monophyletic group with the CDV sequences of other Indian dogs and the Indian wildlife (Lion) and is clustered away from the vaccine strain. The CDV sequences were closer to the CDVs of Africa - 2 lineage than the other Asian lineages. The Recombination analysis revealed no potential breakpoints and recombination events.
 Conclusion: Together, these findings highlighted the possible existence of the novel Indian CDV lineage/s distinct from the vaccine strain and from other known Asian lineages.

Inferred duration of infectious period of SARS-CoV-2: rapid scoping review and analysis of available evidence for asymptomatic and symptomatic COVID-19 cases

ObjectivesOur objective was to review the literature on the inferred duration of the infectious period of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and provide an...

Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids.

Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and β were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and β. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.

The Role of TMPRSS2 and TMPRSS2- Inhibitors in Cell Entry Mechanism of COVID-19

The enzymes trypsin, furine and other proprotein convertases, cathepsin, transmembrane proteases (TMPRSS) and elastases play a role in the cell entry of coronaviruses (Coronaviridae). The proteases TMPRSS2 and TMPRSS11a, which are abundant in the respiratory tract and expressed on cell surfaces, promote the entry of SARS-CoV-1 viruses. For the TMPRSS protease TMPRSS11d - also known as human airway trypsin-like protease (HAT) - a proteolytic activation of the S- protein of SARS-CoV-1 was demonstrated. TMPRSS2, in turn, complexes with the ACE2 receptor, which allows efficient penetration of the virus directly at the cell surface. TMPRSS2 and TMPRSS11D activate the S protein by cleaving it into the S1 and S2 subunits, thus allowing endosome-independent cell entry at the cell membrane. Virus-based therapies include monoclonal antibodies, antiviral peptides that dock to the S protein of viruses, viral nucleic acid synthesis inhibitors and inhibitors for docking to other viral structures and accessory proteins.

Synthesis and evaluation of potent human immunodeficiency virus 1 protease inhibitors with epimeric isopropanol as novel P1' ligands.

Aim: HIV-1 protease inhibitors regimens suffered from a number of drawbacks, among which, the most egregious issue was the growing emergence of drug-resistant strains. Materials & methods: The design strategy of maximizing the protease active site interactions with the inhibitor, especially promoting extensive hydrogen bonding with the protein backbone atoms, might be in favor of combating drug resistance. A series of HIV-1 protease inhibitors that incorporated enantiomeric isopropanols as the P1' ligands in combination with phenols as the P2 ligands were reported herein. Results: A number of inhibitors displayed potent protease enzyme inhibition activity. In particular, inhibitor 14c showed comparable potency as darunavir with IC50 value of 1.91nM and activity against darunavir-resistant HIV-1 variants. Conclusion: The new kind of HIV-1 protease inhibitors deserves further study.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63.

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as β-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC50 = 0.64 μg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC50 values against virus yield were 1.52 μM and 2.60 μM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC50 = 0.06 μM) and indigodole B (IC50 = 2.09 μM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

Y-Box-Binding Protein 3 (YBX3) Restricts Influenza A Virus by Interacting with Viral Ribonucleoprotein Complex and Imparing its Function.

The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.

Targeting viral genome synthesis as broad-spectrum approach against RNA virus infections.

Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the “one drug-multiple bugs”-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes’ universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.

Effective estimation of the inhibitor affinity of HIV-1 protease via a modified LIE approach.

The inhibition of the Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR) can prevent the synthesis of new viruses. Computer-aided drug design (CADD) would enhance the discovery of new therapies, through which the estimation of ligand-binding affinity is critical to predict the most efficient inhibitor. A time-consuming binding free energy method would reduce the usefulness of CADD. The modified linear interaction energy (LIE) approach emerges as an appropriate protocol that performs this task. In particular, the polar interaction free energy, which is obtained via numerically resolving the linear Poisson–Boltzmann equation, plays as an important role in driving the binding mechanism of the HIV-1 PR + inhibitor complex. The electrostatic interaction energy contributes to the attraction between two molecules, but the vdW interaction acts as a repulsive factor between the ligand and the HIV-1 PR. Moreover, the ligands were found to adopt a very strong hydrophobic interaction with the HIV-1 PR. Furthermore, the results obtained corroborate the high accuracy and precision of computational studies with a large correlation coefficient value R = 0.83 and a small RMSE δRMSE = 1.25 kcal mol−1. This method is less time-consuming than the other end-point methods, such as the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) and free energy perturbation (FEP) approaches. Overall, the modified LIE approach would provide ligand-binding affinity with HIV-1 PR accurately, precisely, and rapidly, resulting in a more efficient design of new inhibitors.

Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2–4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 μM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.

Adenoviral strategies to overcome innate cellular responses to infection.

Viruses alter host cell processes to optimize their replication cycle. Human adenoviruses (Ad) encode proteins that promote viral macromolecular synthesis and counteract innate and adaptive responses to infection. The focus of this review is on how Ad evades innate cellular responses to infection, including an interferon (IFN) response and a DNA damage response (DDR). Ad blocks the IFN response by inhibiting cytoplasmic signaling pathways and the activation of IFN-stimulated genes (ISGs), as well as the functions of ISG products, such as PML. Ad also inhibits DDR sensors, for instance, the Mre11-Rad50-Nbs1 complex, and DDR effectors like DNA ligase IV. These innate cellular responses impact many different viruses, and studies on Ad have provided broad insight into these areas.

Zika Virus Dissemination from the Midgut of Aedes aegypti is Facilitated by Bloodmeal-Mediated Structural Modification of the Midgut Basal Lamina.

The arboviral disease cycle requires that key tissues in the arthropod vector become persistently infected with the virus. The midgut is the first organ in the mosquito that needs to be productively infected with an orally acquired virus. Following midgut infection, the virus then disseminates to secondary tissues including the salivary glands. Once these are productively infected, the mosquito is able to transmit the virus to a vertebrate host. Recently, we described the midgut dissemination pattern for chikungunya virus in Aedes aegypti. Here we assess the dissemination pattern in the same mosquito species for Zika virus (ZIKV), a human pathogenic virus belonging to the Flaviviridae. ZIKV infection of secondary tissues, indicative of dissemination from the midgut, was not observed before 72 h post infectious bloodmeal (pibm). Virion accumulation at the midgut basal lamina (BL) was only sporadic, although at 96–120 h pibm, virions were frequently observed between strands of the BL indicative of their dissemination. Our data suggest that ZIKV dissemination from the mosquito midgut occurs after digestion of the bloodmeal. Using gold-nanoparticles of 5 nm and 50 nm size, we show that meal ingestion leads to severe midgut tissue distention, causing the mesh width of the BL to remain enlarged after complete digestion of the meal. This could explain how ZIKV can exit the midgut via the BL after bloodmeal digestion. Ingestion of a subsequent, non-infectious bloodmeal five days after acquisition of an initial, dengue 4 virus containing bloodmeal resulted in an increased number of virions present in the midgut epithelium adjacent to the BL. Thus, subsequent bloodmeal ingestion by an infected mosquito may primarily stimulate de novo synthesis of virions leading to increased viral titers in the vector.

Structure of RdRps Within a Transcribing dsRNA Virus Provides Insights Into the Mechanisms of RNA Synthesis

RNA-dependent RNA polymerases (RdRps) catalyze RNA synthesis of RNA viruses. During initiation of RNA synthesis, the RdRp catalyzes the formation of the first dinucleotide, acting as primer for subsequent processive RNA elongation. Here, we present the structure of the RdRp complexes in the dinucleotide primed state in situ within a transcribing cypovirus under near physiological conditions using cryo-electron microscopy. The 3′ end of RNA templates, paired RNA dinucleotide primer, incoming nucleotide, and catalytic divalent cations in the RdRp were resolved at 3.8 Å resolution. The end of the RNA template and the dinucleotide is buttressed by the aromatic tyrosine in a loop from the RdRp bracelet domain. Our structure reveals the interactions between the nucleotide substrates and the conserved residues during the RdRp initiation, and the coordinated structural changes preceding the elongation stage. In addition, it provides the direct evidence for existence of the slow step of the dinucleotide primed state in the viral RdRp transcription.