Function Of Viral Proteins Research Articles

Recent advances in rotavirus reverse genetics and its utilization in basic research and vaccine development.

Rotaviruses are segmented double-stranded RNA viruses with a high frequency of gene reassortment, and they are a leading cause of global diarrheal deaths in children less than 5 years old. Two-thirds of rotavirus-associated deaths occur in low-income countries. Currently, the available vaccines in developing countries have lower efficacy in children than those in developed countries. Due to added safety concerns and the high cost of current vaccines, there is a need to develop cost-effective next-generation vaccines with improved safety and efficacy. The reverse genetics system (RGS) is a powerful tool for investigating viral protein functions and developing novel vaccines. Recently, an entirely plasmid-based RGS has been developed for several rotaviruses, and this technological advancement has significantly facilitated novel rotavirus research. Here, we review the recently developed RGS platform and discuss its application in studying infection biology, gene reassortment, and development of vaccines against rotavirus disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05142-7.

Reverse genetic engineering of simian rotaviruses with temperature-sensitive lesions in VP1, VP2, and VP6

Rotaviruses are 11-segmented double-stranded RNA viruses and important causes of acute gastroenteritis in young children. To investigate the functions of specific viral proteins during the rotavirus lifecycle, temperature-sensitive (ts) mutants were previously created using a cultivatable simian strain (SA11) and chemical mutagenesis. These ts SA11 mutants replicate more efficiently at the permissive temperature of 31 °C than at the non-permissive temperature of 39 °C. Prototype strains SA11-tsC, SA11-tsF, and SA11-tsG were mapped to the genes encoding structural proteins VP1, VP2, and VP6, respectively, and putative ts lesions were identified using Sanger sequencing. However, additional background mutations in their genomes had hampered validation of the ts lesions and confounded their use in mechanistic studies. Here, we employed plasmid only-based reverse genetics to engineer recombinant (r) SA11 rotaviruses containing only the putative ts lesions of SA11-tsC (L138P change in VP1), SA11-tsF (A387D change in VP2) or SA11-tsG (S10T, D13H, and A121G changes in VP6). For simplicity, we refer to these newly-engineered, isogenic viruses as rSA11-tsVP1, rSA11-tsVP2, and rSA11-tsVP6. Single-cycle growth assays revealed that these mutants indeed exhibit ts phenotypes with significantly diminished titers (>1.5-logs) at 39 °C versus 31 °C. The rSA11 ts mutants proved genetically stable at the population-level following 3 sequential passages at 39 °C, but individual revertant clones were detected in plaque assays. Heat sensitivity experiments showed that pre-incubation of rSA11-tsVP1 or rSA11-tsVP2, but not rSA11-tsVP6, at 39 °C diminished replication at 31 °C. This result indicates that the ts lesions in VP1 and VP2 affect the incoming virion but those in VP6 affect a later stage of the viral lifecycle. In silico molecular dynamics simulations predicted temperature-dependent, long-range effects of the S10T, D13H, and/or A121G changes on the VP6 structure. Altogether, our results confirm the ts lesions of the original SA11-tsC, SA11-tsF, and SA11-tsG mutants, provide a new set of isogenic strains for investigating aspects of rotavirus replication, and shed light on how the ts lesions might impact VP1, VP2, or VP6 functions.

Efam: an expanded, metaproteome-supported HMM profile database of viral protein families.

MotivationViruses infect, reprogram and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases.ResultsHere, we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240 311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, pan-ecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from ‘conservative’ to ‘eXtremely Conservative’ resulted in 37 841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by ∼24% on average (up to ∼42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem.Availability and implementationThe resources are available on the iVirus platform at (doi.org/10.25739/9vze-4143).Supplementary information Supplementary data are available at Bioinformatics online.

Binding mode characterization of 13b in the monomeric and dimeric states of SARS-CoV-2 main protease using molecular dynamics simulations

The main protease, Mpro/3CLpro, plays an essential role in processing polyproteins translated from viral RNA to produce functional viral proteins and therefore serve as an attractive target for discovering COVID-19 therapeutics. The availability of both monomer and dimer crystal bound with a common ligand, ‘13b’ (α-ketoamide inhibitor), opened up opportunities to understand the Mpro mechanism of action. A comparative analysis of both forms of Mpro was carried out to elucidate the binding site architectural differences in the presence and absence of ‘13b’. Molecular dynamics simulations suggest that the presence of ‘13b’ enhances the stability of Mpro than the unbound APO form. The N- and C- terminals of both the protomers stabilize each other, and making it's interface essential for the active form of Mpro. In comparison to monomer, the relatively high affinity of ‘13b’ is gained in dimer pocket due to the high stability of the pocket by the interaction of S1 residue of chain B with residues F140, E166 and H172 of chain A, which is absent in monomer. The comprehensive essential dynamics, protein structure network analysis and thermodynamic profiling highlight the hot-spots, pivotal in molecular recognition process at protein-ligand and protein-protein interaction levels, cross-validated through computational alanine scanning study. A comparative description of ‘13b’ binding mechanism in both forms illustrates valuable insights into the inhibition mechanism and the selection of critical residues suitable for the structure-based approaches for the identification of more potent Mpro inhibitors. Communicated by Ramaswamy H. Sarma

A large-scale screen to target SARS-CoV-2

Coronavirus The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, Mpro, is essential to yield functional viral proteins, making it a key drug target. Gunther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to Mpro. High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science , this issue p. [642][1] [1]: /lookup/doi/10.1126/science.abf7945

Identification, Characterization and Drug Discovery for Novel Target Sites for SARS‐CoV‐2 Proteins

The virus SARS‐CoV‐2, the cause of the current COVID‐19 pandemic, is not well understood. It is critical to understand how the viral proteins function and how their function may be modulated. Inhibitors that target these enzymes serve as potential therapeutic interventions against COVID‐19. This work uses artificial intelligence methods developed by us to find sites that other methods may not find and therefore, identify potential exosites, allosteric sites, or other sites of interaction in the structures of viral proteins to serve as new targets for the development of antiviral agents. Large datasets of natural and synthetic compounds are computationally searched for molecules that fit into these alternative sites, and any compounds that fit will be targeted for experimental testing for their ability to inhibit the functions of these viral enzymes. This project uses the unique Partial Order Optimum Likelihood (POOL) machine learning method developed by us to predict multiple types of binding sites in SARS‐CoV‐2 proteins, including catalytic sites, allosteric sites, and other interaction sites. Molecular dynamics simulations are used to generate conformations for ensemble docking. Compounds from large molecular libraries are computationally docked into the predicted sites to identify potentially strong binding ligands. We have identified approximately 10000 potential ligands for more than 50 SARS‐CoV‐2 proteins to date. Candidate ligands to selected SARS‐CoV‐2 proteins are experimentally tested in vitro for binding affinity and the effect of the best‐predicted inhibitors on catalytic activities determined by direct biochemical assays. Compound libraries for the study include selected compounds from the ZINC and Enamine databases; Chemical Service database compounds and COVID‐specific libraries from Enamine and Life Chemicals.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Targeting the SARS-CoV-2 main protease

Coronavirus Vaccines are an important tool in the fight against COVID-19, but developing antiviral drugs is also a high priority, especially with the rise of variants that may partially evade vaccines. The viral protein main protease is required for cleaving precursor polyproteins into functional viral proteins. This essential function makes it a key drug target. Qiao et al. designed 32 inhibitors based on either boceprevir or telaprevir, both of which are protease inhibitors approved to treat hepatitis C virus. Six compounds protected cells from viral infection with high potency, and two of these were selected for in vivo studies based on pharmokinetic experiments. Both showed strong antiviral activity in a mouse model. Science , this issue p. [1374][1] [1]: /lookup/doi/10.1126/science.abf1611

TMBIM6, a potential virus target protein identified by integrated multiomics data analysis in SARS-CoV-2-infected host cells.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we collected open access data to analyze the mechanisms associated with SARS-CoV-2 infection. Gene set enrichment analysis (GSEA) revealed that apoptosis-related pathways were enriched in the cells after SARS-CoV-2 infection, and the results of differential expression analysis showed that biological functions related to endoplasmic reticulum stress (ERS) and lipid metabolism were disordered. TMBIM6 was identified as a potential target for SARS-CoV-2 in host cells through weighted gene coexpression network analysis (WGCNA) of the time course of expression of host and viral proteins. The expression and related functions of TMBIM6 were subsequently analyzed to illuminate how viral proteins interfere with the physiological function of host cells. The potential function of viral proteins was further analyzed by GEne Network Inference with Ensemble of trees (GENIE3). This study identified TMBIM6 as a target protein associated with the pathogenesis of SARS-CoV-2, which might provide a novel therapeutic approach for COVID-19 in the future.

The Missing Expression Level-Evolutionary Rate Anticorrelation in Viruses Does Not Support Protein Function as a Main Constraint on Sequence Evolution.

One of the central goals in molecular evolutionary biology is to determine the sources of variation in the rate of sequence evolution among proteins. Gene expression level is widely accepted as the primary determinant of protein evolutionary rate, because it scales with the extent of selective constraints imposed on a protein, leading to the well-known negative correlation between expression level and protein evolutionary rate (the E–R anticorrelation). Selective constraints have been hypothesized to entail the maintenance of protein function, the avoidance of cytotoxicity caused by protein misfolding or nonspecific protein–protein interactions, or both. However, empirical tests evaluating the relative importance of these hypotheses remain scarce, likely due to the nontrivial difficulties in distinguishing the effect of a deleterious mutation on a protein’s function versus its cytotoxicity. We realized that examining the sequence evolution of viral proteins could overcome this hurdle. It is because purifying selection against mutations in a viral protein that result in cytotoxicity per se is likely relaxed, whereas purifying selection against mutations that impair viral protein function persists. Multiple analyses of SARS-CoV-2 and nine other virus species revealed a complete absence of any E–R anticorrelation. As a control, the E–R anticorrelation does exist in human endogenous retroviruses where purifying selection against cytotoxicity is present. Taken together, these observations do not support the maintenance of protein function as the main constraint on protein sequence evolution in cellular organisms.

SARS-CoV-2 M pro inhibitors with antiviral activity in a transgenic mouse model

The COVID-19 pandemic caused by the SARS-CoV-2 virus continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either Boceprevir or Telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro with IC50 values ranging from 7.6 to 748.5 nM. The co-crystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a SARS-CoV-2 infection transgenic mouse model, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

A comprehensive profile of genomic variations in the SARS-CoV-2 isolates from the state of Telangana, India.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 has rapidly turned into a pandemic, infecting millions and causing 1 157 509 (as of 27 October 2020) deaths across the globe. In addition to studying the mode of transmission and evasion of host immune system, analysing the viral mutational landscape constitutes an area under active research. The latter is expected to impart knowledge on the emergence of different clades, subclades, viral protein functions and protein–protein and protein–RNA interactions during replication/transcription cycle of virus and response to host immune checkpoints. In this study, we have attempted to bring forth the viral genomic variants defining the major clade(s) as identified from samples collected from the state of Telangana, India. We further report a comprehensive draft of all genomic variations (including unique mutations) present in SARS-CoV-2 strain in the state of Telangana. Our results reveal the presence of two mutually exclusive subgroups defined by specific variants within the dominant clade present in the population. This work attempts to bridge the critical gap regarding the genomic landscape and associate mutations in SARS-CoV-2 from a highly infected southern region of India, which was lacking to date.

Prediction and Analysis of Multiple Sites and Inhibitors of SARS-CoV-2 Proteins

In the current COVID-19 pandemic, it is critical to understand, as swiftly as possible, how the viral proteins function and how their function might be modulated. The machine learning method Partial Order Optimum Likelihood (POOL) is used to predict binding sites in protein structures from SARS-CoV-2, the virus that causes COVID-19. Using the 3D structure of each protein as input, POOL uses computed electrostatic and chemical properties to predict the amino acids that are biochemically active, including residues in catalytic sites, allosteric sites, and other secondary sites.

Virus-Encoded Complement Regulators: Current Status.

Viruses require a host for replication and survival and hence are subjected to host immunological pressures. The complement system, a crucial first response of the host immune system, is effective in targeting viruses and virus-infected cells, and boosting the antiviral innate and acquired immune responses. Thus, the system imposes a strong selection pressure on viruses. Consequently, viruses have evolved multiple countermeasures against host complement. A major mechanism employed by viruses to subvert the complement system is encoding proteins that target complement. Since viruses have limited genome size, most of these proteins are multifunctional in nature. In this review, we provide up to date information on the structure and complement regulatory functions of various viral proteins.

The Merkel Cell Polyomavirus T Antigens Function as Tumor Promoters in Murine Skin.

Simple SummaryMerkel cell polyomavirus, a recently discovered human virus, is linked to the development of a rare form of skin cancer called Merkel cell carcinoma. The virus does not replicate in cancer cells, yet there is continued expression of viral proteins known as T antigens. The T antigens are believed to contribute to Merkel cell carcinoma development, yet how they do so remains an active area of research. In this study, we used transgenic mice expressing the viral T antigens in their skin to determine at which stage of skin cancer development these viral proteins function. We discovered that the Merkel cell polyomavirus T antigens function as tumor promoters, rather than tumor initiators, in the skin. These findings suggest that other tumor-initiating events may cooperate with the tumor-promoting activities of the viral T antigens, thus providing important insight into how Merkel cell polyomavirus can cause cancer in human skin.Merkel cell polyomavirus (MCPyV) causes the majority of human Merkel cell carcinomas (MCC), a rare but highly aggressive form of skin cancer. We recently reported that constitutive expression of MCC tumor-derived MCPyV tumor (T) antigens in the skin of transgenic mice leads to hyperplasia, increased proliferation, and spontaneous epithelial tumor development. We sought to evaluate how the MCPyV T antigens contribute to tumor formation in vivo using a classical, multi-stage model for squamous cell carcinoma development. In this model, two chemical carcinogens, DMBA and TPA, contribute to two distinct phases of carcinogenesis—initiation and promotion, respectively—that are required for tumors to develop. By treating the MCPyV transgenic mice with each chemical carcinogen, we determined how the viral oncogenes contributed to carcinogenesis. We observed that the MCPyV T antigens synergized with the tumor initiator DMBA, but not with the tumor promoter TPA, cause tumors. Therefore, the MCPyV tumor antigens function primarily as tumor promoters, similar to that seen with human papillomavirus (HPV) oncoproteins. These studies provide insight into the role of MCPyV T antigen expression in tumor formation in vivo and contribute to our understanding of how MCPyV may function as a human DNA tumor virus.

Genes of SARS-CoV-2 and emerging variants

The pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is distinctly different from outbreaks caused by other coronaviruses: SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The differences in the rapid transmission and severity of human coronaviruses are due to the genetic composition of the virus. SARS-CoV-2 contains genes encoding non-structural proteins (NSPs), structural proteins, and accessory proteins. The NSPs are mainly involved in replication of the virus within the host and inhibition of the host defence system. Structural proteins are involved in viral entry and attachment to host cells, preservation of the core virion and elicit the majority of the immune response. The functions of the accessory proteins are largely unknown. Most focus has been given to structural proteins, especially the spike protein as the strongest vaccine candidate. However, the recent emergence of spike variants and their ability to rapidly transmit and escape neutralisation by vaccine-induced antibodies has threatened the global community. Meanwhile, recent studies of accessory proteins reveal their importance in viral pathogenesis. Hence, proper understanding of the functions of all unknown viral proteins is crucial to devise alternate antiviral strategies.

Dual targeting of 3CLpro and PLpro of SARS-CoV-2: A novel structure-based design approach to treat COVID-19.

With the rapid growth of the COVID-19 (coronavirus disease 2019) pandemic across the globe, therapeutic attention must be directed to fight the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). However, developing new antiviral drugs and vaccines is time-consuming, so one of the best solutions to tackle this virus at present is to repurpose ready-to-use drugs. This paper proposes the repurposing of the Food and Drug Administration (FDA)-approved, purchasable, and naturally occurring drugs for preventive and therapeutic use. We propose to design a dual-inhibitor for the SARS-CoV-2 cysteine proteases—3 Chemotrypsin-like protease or main protease (3CLpro or Mpro) and Papain-like protease (PLpro) responsible for processing the translated polyprotein chain from the viral RNA yielding functional viral proteins. For virtual screening, an unbiased blind docking was performed from which the top nine dual-targeting inhibitors for 3CLpro and PLpro were selected. The nine repurposed drugs, block the catalytic dyad (His41 and Cys145) of 3CLpro as well as the catalytic triad (Cys111, His272, and Asp286) of PLpro. Repurposing known drugs will not only pave the way for rapid in-vitro and in-vivo studies to battle the SARS-CoV-2 but will also expedite the quest for a potent anti-coronaviral drug.

Hepatitis B Core Protein Is Post-Translationally Modified through K29-Linked Ubiquitination

Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

Hantavirus Infection Is Inhibited by Griffithsin in Cell Culture.

Andes virus (ANDV) and Sin Nombre virus (SNV), highly pathogenic hantaviruses, cause hantavirus pulmonary syndrome in the Americas. Currently no therapeutics are approved for use against these infections. Griffithsin (GRFT) is a high-mannose oligosaccharide-binding lectin currently being evaluated in phase I clinical trials as a topical microbicide for the prevention of human immunodeficiency virus (HIV-1) infection (ClinicalTrials.gov Identifiers: NCT04032717, NCT02875119) and has shown broad-spectrum in vivo activity against other viruses, including severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, and Nipah virus. In this study, we evaluated the in vitro antiviral activity of GRFT and its synthetic trimeric tandemer 3mGRFT against ANDV and SNV. Our results demonstrate that GRFT is a potent inhibitor of ANDV infection. GRFT inhibited entry of pseudo-particles typed with ANDV envelope glycoprotein into host cells, suggesting that it inhibits viral envelope protein function during entry. 3mGRFT is more potent than GRFT against ANDV and SNV infection. Our results warrant the testing of GRFT and 3mGRFT against ANDV infection in animal models.