Identification of cellular proteins associated with human cytomegalovirus (HCMV) DNA replication suggests novel cellular and viral interactions.

Upon entry of Human cytomegalovirus (HCMV) into the host cell, the viral genome is transported to the nucleus where it serves as a template for transcription and genome replication. Production of new viral genomes is a coordinated effort between viral and cellular proteins. While the core replication proteins are encoded by the virus, additional cellular proteins support the process of genome synthesis. We used accelerated native isolation of proteins on nascent DNA (aniPOND) to study protein dynamics on nascent viral DNA during HCMV infection. Using this method, we identified specific viral and cellular proteins that are associated with nascent viral DNA. These included transcription factors, transcriptional regulators, DNA damage and repair factors, and chromatin remodeling complexes. The association of these identified proteins with viral DNA was confirmed by immunofluorescent imaging, chromatin-immunoprecipitation analyses, and shRNA knockdown experiments. These data provide evidence for the requirement of cellular factors involved in HCMV replication.

Picornaviruses: A View from 3A.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

Uncovering potential host proteins and pathways that may interact with eukaryotic short linear motifs in viral proteins of MERS, SARS and SARS2 coronaviruses that infect humans.

A coronavirus pandemic caused by a novel coronavirus (SARS-CoV-2) has spread rapidly worldwide since December 2019. Improved understanding and new strategies to cope with novel coronaviruses are urgently needed. Viruses (especially RNA viruses) encode a limited number and size (length of polypeptide chain) of viral proteins and must interact with the host cell components to control (hijack) the host cell machinery. To achieve this goal, the extensive mimicry of SLiMs in host proteins provides an effective strategy. However, little is known regarding SLiMs in coronavirus proteins and their potential targets in host cells. The objective of this study is to uncover SLiMs in coronavirus proteins that are present within host cells. These SLiMs have a high possibility of interacting with host intracellular proteins and hijacking the host cell machinery for virus replication and dissemination. In total, 1,479 SLiM hits were identified in the 16 proteins of 590 coronaviruses infecting humans. Overall, 106 host proteins were identified that may interact with SLiMs in 16 coronavirus proteins. These SLiM-interacting proteins are composed of many intracellular key regulators, such as receptors, transcription factors and kinases, and may have important contributions to virus replication, immune evasion and viral pathogenesis. A total of 209 pathways containing proteins that may interact with SLiMs in coronavirus proteins were identified. This study uncovers potential mechanisms by which coronaviruses hijack the host cell machinery. These results provide potential therapeutic targets for viral infections.

Investigation of genetic variation: Neuraminidase gene of influenza A virus H1N1/pdm09, Shiraz, Iran (2015-2016).

Oseltamivir and antiviral agents are frequently used for the prevention and treatment of influenza infection. However, resistance to oseltamivir has been reported globally due to a mutation in the Influenza virus neuraminidase gene. Such resistance will be detected by genotyping and phenotyping studies of viral isolates. The recent study aimed to determine the genetic mutation of neuraminidase gene in influenza A (H1N1) viruses isolated from children referred to Shiraz tertiary hospitals during 1 year (2015-2016) with influenza-like symptoms. A total of 300 patients were registered and throat samples were taken. The throat swabs were used for viral RNA extraction. Detection of influenza A (H1N1) was performed using the one-step real-time polymerase chain reaction (qRT-PCR) method. From positive isolates for H1N1, 51 random samples were evaluated for neuraminidase gene mutation with the nested PCR-sequencing method. Of 300 cases, 102 (34%) isolates were detected as influenza A (H1N1) pdm09. Based on sequencing results, 2 of the 44 sequenced isolates exhibited H275Y substitution, which presented oseltamivir resistance. In comparison with reference strain, the phylogenetic analysis of sequenced isolates was classified in genogroup 6B. While this result is the first report of emerging oseltamivir-resistant in the southwest of Iran, it is highly recommended to perform these evaluations on the different geographical regions in any prevalence area to plan treatment strategies for influenza.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

Classification and characterization of multigene family proteins of African swine fever viruses.

African swine fever virus (ASFV) poses serious threats to the pig industry. The multigene family (MGF) proteins are extensively distributed in ASFVs and are generally classified into five families, including MGF-100, MGF-110, MGF-300, MGF-360 and MGF-505. Most MGF proteins, however, have not been well characterized and classified within each family. To bridge this gap, this study first classified MGF proteins into 31 groups based on protein sequence homology and network clustering. A web server for classifying MGF proteins was established and kept available for free at http://www.computationalbiology.cn/MGF/home.html. Results showed that MGF groups of the same family were most similar to each other and had conserved sequence motifs; the genetic diversity of MGF groups varied widely, mainly due to the occurrence of indels. In addition, the MGF proteins were predicted to have large structural and functional diversity, and MGF proteins of the same MGF family tended to have similar structure, location and function. Reconstruction of the ancestral states of MGF groups along the ASFV phylogeny showed that most MGF groups experienced either the copy number variations or the gain-or-loss changes, and most of these changes happened within strains of the same genotype. It is found that the copy number decrease and the loss of MGF groups were much larger than the copy number increase and the gain of MGF groups, respectively, suggesting the ASFV tended to lose MGF proteins in the evolution. Overall, the work provides a detailed classification for MGF proteins and would facilitate further research on MGF proteins.

METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies-Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses.

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.

Intrinsically disordered proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis.

Intrinsically disordered proteins (IDPs) possess the property of inherent flexibility and can be distinguished from other proteins in terms of lack of any fixed structure. Such dynamic behavior of IDPs earned the name "Dancing Proteins." The exploration of these dancing proteins in viruses has just started and crucial details such as correlation of rapid evolution, high rate of mutation and accumulation of disordered contents in viral proteome at least understood partially. In order to gain a complete understanding of this correlation, there is a need to decipher the complexity of viral mediated cell hijacking and pathogenesis in the host organism. Further there is necessity to identify the specific patterns within viral and host IDPs such as aggregation; Molecular recognition features (MoRFs) and their association to virulence, host range and rate of evolution of viruses in order to tackle the viral-mediated diseases. The current book chapter summarizes the aforementioned details and suggests the novel opportunities for further research of IDPs senses in viruses.

Amino acid sequence associated with bacteriophage recombination site helps to reveal genes potentially acquired through horizontal gene transfer.

BACKGROUND: Horizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer. RESULTS: HMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events. CONCLUSIONS: Cysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing.

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.

Overwhelming mutations or SNPs of SARS-CoV-2: A point of caution.

The morbidity of SARS-CoV-2 (COVID-19) is reaching 3 Million landmark causing and a serious public health concern globally and it is enigmatic how several antiviral and antibody treatments were not effective in the different period across the globe. With the drastic increasing number of positive cases around the world WHO raised the importance in the assessment of the risk of spread and understanding genetic modifications that could have occurred in the SARS-CoV-2. Using all available deep sequencing data of complete genome from all over the world (NCBI repository), we identified several hundreds of point mutations or SNPs in SARS-CoV-2 all across the genome. This could be the cause for the constant change and differed virulence with an increase in mortality and morbidity. Among the 12 different countries (one sequence from each country) with complete genome sequencing data, we noted the 47 key point mutations or SNPs located along the entire genome that might have impact in the virulence and response to different antivirals against SARS-CoV-2. In this regard, key viral proteins of spike glycoprotein, Nsp1, RdRp and the ORF8 region got heavily mutated within these 3 months via person-to-person passage. We also discuss what could be the possible cause of this rapid mutation in the SARS-CoV-2.

Deconvolving mutational patterns of poliovirus outbreaks reveals its intrinsic fitness landscape.

Vaccination has essentially eradicated poliovirus. Yet, its mutation rate is higher than that of viruses like HIV, for which no effective vaccine exists. To investigate this, we infer a fitness model for the poliovirus viral protein 1 (vp1), which successfully predicts in vitro fitness measurements. This is achieved by first developing a probabilistic model for the prevalence of vp1 sequences that enables us to isolate and remove data that are subject to strong vaccine-derived biases. The intrinsic fitness constraints derived for vp1, a capsid protein subject to antibody responses, are compared with those of analogous HIV proteins. We find that vp1 evolution is subject to tighter constraints, limiting its ability to evade vaccine-induced immune responses. Our analysis also indicates that circulating poliovirus strains in unimmunized populations serve as a reservoir that can seed outbreaks in spatio-temporally localized sub-optimally immunized populations.

An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Mumps: MMR vaccination and genetic diversity of mumps virus, 2007-2011 in Catalonia, Spain.

BACKGROUND: Mumps is a vaccine-preventable disease but outbreaks have been reported in persons vaccinated with two doses of MMR vaccine. The objective was to describe the demographic features, vaccination effectiveness and genetic mumps virus diversity among laboratory-confirmed cases between 2007 and 2011 in Catalonia. METHODS: Cases and outbreaks of mumps notified to the notifiable diseases system of Catalonia between 2007 and 2011 retrospectively registered were included. Public health care centres provided written immunization records to regional public health staff to determine the vaccination history. Saliva and serum specimens were collected from suspected cases for laboratory-confirmation using real-time reverse-transcriptase PCR (rtRT-PCR) or serological testing. Phylogenetic analysis of the complete SH gene (316 nucleotides) and complete coding HN protein (1749 nucleotides) sequences was made. Categorical variables were compared using the Chi-square or Fisher's tests and continuous variables using the Student test. Vaccination effectiveness by number of MMR doses was estimated using the screening method. RESULTS: During the study period, 581 confirmed cases of mumps were notified (incidence rate 1.6 cases/100,000 persons-year), of which 60% were male. Three hundred sixty-four laboratory-confirmed cases were reported, of which 44% were confirmed by rtRT-PCR. Of the 289 laboratory-confirmed cases belonging to vaccination cohorts, 33.5% (97) had received one dose of MMR vaccine and 50% (145) two doses. Based on phylogenetic analyses of 316-nucleotide and 174-nucleotide SH sequences, the viruses belonging to viral genotypes were: genotype G (126), genotype D (23), genotype H (2), genotype F (2), genotype J (1), while one remained uncharacterized. Amino acid differences were detected between circulating strains and the Jeryl Lynn vaccine strains, although the majority of amino acid substitutions were genotype-specific. Fifty-one outbreaks were notified that included 324 confirmed mumps cases. Genotype G was the most frequent genotype detected. The family (35%), secondary schools (25%) and community outbreaks (18%) were the most frequent settings. CONCLUSIONS: Our study shows that genotype G viruses are the most prevalent in Catalonia. Most cases occurred in people who had received two doses of MMR, suggesting inadequate effectiveness of the Jeryl Lynn vaccine strain. The possible factors related are discussed.

A novel group of avian astroviruses from Neotropical passerine birds broaden the diversity and host range of Astroviridae.

Metagenomics is helping to expand the known diversity of viruses, especially of those with poorly studied hosts in remote areas. The Neotropical region harbors a considerable diversity of avian species that may play a role as both host and short-distance vectors of unknown viruses. Viral metagenomics of cloacal swabs from 50 Neotropical birds collected in French Guiana revealed the presence of four complete astrovirus genomes. They constitute an early diverging novel monophyletic clade within the Avastrovirus phylogeny, representing a putative new astrovirus species (provisionally designated as Avastrovirus 5) according to the International Committee on Taxonomy of Viruses (ICTV) classification criteria. Their genomic organization shares some characteristics with Avastrovirus but also with Mamastrovirus. The pan-astrovirus RT-PCR analysis of the cloacal samples of 406 wild Neotropical birds showed a community-level prevalence of 4.9% (5.1% in passerines, the highest described so far in this order of birds). By screening birds of a remote region, we expanded the known host range of astroviruses to the avian families Cardinalidae, Conopophagidae, Furnariidae, Thamnophilidae, Turdidae and Tyrannidae. Our results provide important first insights into the unexplored viral communities, the ecology, epidemiology and features of host-pathogen interactions that shape the evolution of avastroviruses in a remote Neotropical rainforest.

Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.

Comprehensive mapping of adaptation of the avian influenza polymerase protein PB2 to humans.

Viruses like influenza are infamous for their ability to adapt to new hosts. Retrospective studies of natural zoonoses and passaging in the lab have identified a modest number of host-adaptive mutations. However, it is unclear if these mutations represent all ways that influenza can adapt to a new host. Here we take a prospective approach to this question by completely mapping amino-acid mutations to the avian influenza virus polymerase protein PB2 that enhance growth in human cells. We identify numerous previously uncharacterized human-adaptive mutations. These mutations cluster on PB2's surface, highlighting potential interfaces with host factors. Some previously uncharacterized adaptive mutations occur in avian-to-human transmission of H7N9 influenza, showing their importance for natural virus evolution. But other adaptive mutations do not occur in nature because they are inaccessible via single-nucleotide mutations. Overall, our work shows how selection at key molecular surfaces combines with evolutionary accessibility to shape viral host adaptation.

Evaluation of a concatenated protein phylogeny for classification of tailed double-stranded DNA viruses belonging to the order Caudovirales.

Viruses of bacteria and archaea are important players in global carbon cycling as well as drivers of host evolution, yet the taxonomic classification of viruses remains a challenge due to their genetic diversity and absence of universally conserved genes. Traditional classification approaches employ a combination of phenotypic and genetic information which is no longer scalable in the era of bulk viral genome recovery through metagenomics. Here, we evaluate a phylogenetic approach for the classification of tailed double-stranded DNA viruses from the order Caudovirales by inferring a phylogeny from the concatenation of 77 single-copy protein markers using a maximum-likelihood method. Our approach is largely consistent with the International Committee on Taxonomy of Viruses, with 72 and 89% congruence at the subfamily and genus levels, respectively. Discrepancies could be attributed to misclassifications and a small number of highly mosaic genera confounding the phylogenetic signal. We also show that confidently resolved nodes in the concatenated protein tree are highly reproducible across different software and models, and conclude that the approach can serve as a framework for a rank-normalized taxonomy of most tailed double-stranded DNA viruses.

Molecular characterization of echovirus 12 strains isolated from healthy children in China.

Human echovirus 12 (E-12) belongs to the enterovirus B species. To date, only one full-length genome sequence of E-12 (prototype strain Travis) is available in the GenBank database. This study determined the complete sequence of three E-12 strains, which were isolated from the stools of three healthy children in Yunnan, China, in 2013. We revealed that the three Yunnan E-12 strains had only 80.8-80.9% nucleotide identity and 96.4-96.8% amino acid identity with the Travis strain based on pairwise comparisons of the complete genome nucleotide and amino acid sequences. The three Yunnan strains shared 99.7% nucleotide identity and 99.1-99.5% amino acid similarity. Phylogenetic and similarity plot analyses showed that intertypic recombination occurred in the non-structural regions of the three Yunnan E-12 strains. This is the first report of the complete genome sequence of E-12 in China and it enriches the complete genome sequences of E-12 in the GenBank database.