Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein.

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus.

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

A comprehensive analysis of SARS-CoV-2 missense mutations indicates that all possible amino acid replacements in the viral proteins occurred within the first two-and-a-half years of the pandemic.

The surveillance of COVID-19 pandemic has led to the determination of millions of genome sequences of the SARS-CoV-2 virus, with the accumulation of a wealth of information never collected before for an infectious disease. Exploring the information retrieved from the GISAID database reporting at that time >13 million genome sequences, we classified the 141,639 unique missense mutations detected in the first two-and-a-half years (up to October 2022) of the pandemic. Notably, our analysis indicates that 98.2 % of all possible conservative amino acid replacements occurred. Even non-conservative mutations were highly represented (73.9 %). For a significant number of residues (3 %), all possible replacements with the other nineteen amino acids have been observed. These observations strongly indicate that, in this time interval, the virus explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain and that those that are not observed severely affect SARS-CoV-2 integrity. The implications of the present findings go well beyond the structural biology of SARS-CoV-2 as the huge amount of information here collected and classified may be valuable for the elucidation of the sequence-structure-function relationships in proteins.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Identification of a leucine-zipper motif in pUL51 essential for HCMV replication and potential target for antiviral development.

Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Use of current antivirals is limited by their adverse effects and emergence of drug resistance mutations. Thus, new drugs are an urgent need. The terminase complex (pUL56-pUL89-pUL51) represents a target of choice for new antivirals development. pUL51 was shown to be crucial for the cleavage of concatemeric HCMV DNA and viral replication. Its C-terminal part plays a critical role for the terminase complex assembly. However, no interaction domain is clearly identified. Sequence comparison of herpesvirus homologs and protein modelling were performed on pUL51. Importance of a putative interaction domain is validated by the generation of recombinant viruses with specific alanine substitutions of amino acids implicated in the domain. We identified a Leucine-Zipper (LZ) domain involving the leucine residues L126-X6-L133-X6-L140-X6-L147 in C-terminal part of pUL51. These leucines are crucial for viral replication, suggesting the significance for pUL51 structure and function. A mimetic-peptide approach has been used and tested in antiviral assays to validate the interaction domain as a new therapeutic target. Cytotoxicity was evaluated by LDH release measurement. The peptide TAT-HK29, homologous to the pUL51-LZ domain, inhibits HCMV replication by 27% ± 9% at 1.25 µM concentration without cytotoxicity. Our results highlight the importance of a leucine zipper domain in the C-terminal part of pUL51 involving leucines L126, L133, L140 and L147. We also confirm the potential of mimetic peptides to inhibit HCMV replication and the importance to target interaction domains to develop antiviral agents.

Investigating the aggregation perspective of Dengue virus proteome.

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the ß-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities.

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data.

The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy-terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610-630.

Molecular characterization of a novel deltaflexivirus infecting the edible fungus Pleurotus ostreatus.

A novel positive single-stranded RNA virus, Pleurotus ostreatus deltaflexivirus 1 (PoDFV1), was isolated from the edible fungus Pleurotus ostreatus strain ZP6. The complete genome of PoDFV1 is 7706 nucleotides (nt) long and contains a short poly(A) tail. PoDFV1 was predicted to contain one large open reading frame (ORF1) and three small downstream ORFs (ORFs 2-4). ORF1 encodes a putative replication-associated polyprotein of 1979 amino acids (aa) containing three conserved domains - viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) - which are common to all deltaflexiviruses. ORFs 2-4 encode three small hypothetical proteins (15-20 kDa) without conserved domains or known biological functions. Sequence alignments and phylogenetic analysis suggested that PoDFV1 is a member of a new species in the genus Deltaflexivirus (family Deltaflexiviridae, order Tymovirales). To our knowledge, this is the first report of a deltaflexivirus infecting P. ostreatus.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

Effect of Passive Administration of Monoclonal Antibodies Recognizing Simian Immunodeficiency Virus (SIV) V2 in CH59-Like Coil/Helical or ß-Sheet Conformations on Time of SIVmac251 Acquisition.

The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a ß-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIVmac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIVmac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14+ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44+ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIVmac251 acquisition, highlighting the complexity of antibody responses to V2.

Molecular characterization of a novel victorivirus isolated from the phytopathogenic fungus Phaeobotryon rhois.

Phaeobotryon rhois is an important pathogenic fungus that causes dieback and canker disease of woody hosts. A novel mycovirus, tentatively named "Phaeobotryon rhois victorivirus 1" (PrVV1), was identified in P. rhois strain SX8-4. The PrVV1 has a double-stranded RNA (dsRNA) genome that is 5,224 base pairs long and contains two open reading frames (ORF1 and ORF2), which overlap at a AUGA sequence. ORF1 encodes a polypeptide of 786 amino acids (aa) that contains the conserved coat protein (CP) domain of victoriviruses, while ORF2, encodes a large polypeptide of 826 aa that contains the conserved RNA-dependent RNA polymerase (RdRp) domain of victoriviruses. Our analysis of genomic structure, homology, and phylogeny indicated that PrVV1 is a novel member of the genus Victorivirus in the family Totiviridae. This is the first report of the complete genome sequence of a victorivirus that infects P. rhois.

Movement Protein Mediates Systemic Necrosis in Tomato Plants with Infection of Tomato Mosaic Virus.

The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants.

Do variations in the HLA-E ligand encoded by UL40 distinguish individuals susceptible to HCMV disease?

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort.

De novo designed EBAI as a potential inhibitor of the viral protein BHRF1. Research in silico.

Epstein-Barr virus is a DNA-containing virus that, according to current data, is associated with approximately 1% of all cancers in the world. This viral effect on the human body is associated with its pronounced antiapoptotic activity. An important role in this process is played by the protein BHRF1, which is a structural and functional homologue of antiapoptotic proteins of the BCL-2 family. In this study, we investigate the selective low molecular weight inhibitor of the above viral protein - EBAI (Epstein-Barr virus Antiapoptotic Inhibitor), which we designed using in silico methods. We conducted two parallel simulation experiments where EBAI was intentionally destabilized to demonstrate its high-affinity recognition potential of the BHRF1 pocket, which binds BH3.Thus, although the potential inhibitor was in close proximity to the site of interaction, it contacted it only through orientation interactions (hydrogen and Coulomb interactions). Despite this complication of the standard ligand-receptor complex simulation procedure, we demonstrated in two parallel computational experiments the high affinity of EBAI for the BH3-binding pocket of BHRF1. In both cases, in the first nanoseconds of modeling, our inhibitor underwent the necessary conformational rearrangements and formed all the required interactions for effective complexation. Thus, further in vitro studies are logical and necessary step to fully evaluate the potential of EBAI as an inhibitor of the antiapoptotic protein BHRF1 of Epstein-Barr virus.Communicated by Ramaswamy H. Sarma.

Mass spectrometry detection of monkeypox virus: Comprehensive coverage for ranking the most responsive peptide markers.

The recent and sudden outbreak of monkeypox in numerous non-endemic countries requires expanding its surveillance immediately and understanding its origin and spread. As learned from the COVID-19 pandemic, appropriate detection techniques are crucial to achieving such a goal. Mass spectrometry has the advantages of a rapid response, low analytical interferences, better precision, and easier multiplexing to detect various pathogens and their variants. In this proteomic dataset, we report experimental data on the proteome of the monkeypox virus (MPXV) recorded by state-of-the-art shotgun proteomics, including data-dependent and data-independent acquisition for comprehensive coverage. We highlighted 152 viral proteins, corresponding to an overall proteome coverage of 79.5 %. Among the 1371 viral peptides detected, 35 peptides with the most intense signals in mass spectrometry were selected, representing a subset of 13 viral proteins. Their relevance as potential candidate markers for virus detection by targeted mass spectrometry is discussed. This report should assist the rapid development of mass spectrometry-based tests to detect a pathogen of increasing concern.

Gold-Based Metal Drugs as Inhibitors of Coronavirus Proteins: The Inhibition of SARS-CoV-2 Main Protease by Auranofin and Its Analogs.

Gold compounds have a long tradition in medicine and offer many opportunities for new therapeutic applications. Herein, we evaluated the lead compound Auranofin and five related gold(I) complexes as possible inhibitors of SARS-CoV-2 Main Protease (SARS-CoV-2 Mpro), a validated drug target for the COVID-19 disease. The investigational panel of gold compounds included Auranofin; three halido analogues, i.e., Au(PEt3)Cl, Au(PEt3)Br, and Au(PEt3)I; and two gold carbene complexes, i.e., Au(NHC)Cl and [Au(NHC)2]PF6. Notably, all these gold compounds, with the only exception of [Au(NHC)2]PF6, turned out to be potent inhibitors of the catalytic activity of SARS-CoV-2 Mpro: the measured Ki values were in the range 2.1-0.4 µM. The reactions of the various gold compounds with SARS-CoV-2 Mpro were subsequently investigated through electrospray ionization (ESI) mass spectrometry (MS) upon a careful optimization of the experimental conditions; the ESI MS spectra provided clear evidence for the formation of tight metallodrug-protein adducts and for the coordination of well defined gold-containing fragments to the SARS-CoV-2 Mpro, again with the only exception of [Au(NHC)2]PF6, The metal-protein stoichiometry was unambiguously determined for the resulting species. The crystal structures of the metallodrug- Mpro adducts were solved in the case of Au(PEt3)Br and Au(NHC)Cl. These crystal structures show that gold coordination occurs at the level of catalytic Cys 145 in the case of Au(NHC)Cl and at the level of both Cys 145 and Cys 156 for Au(PEt3)Br. Tight coordination of gold atoms to functionally relevant cysteine residues is believed to represent the true molecular basis of strong enzyme inhibition.

Characterization of a novel victorivirus from Nigrospora chinensis, a fungus isolated from tobacco.

Here, we characterized a new mycovirus from the fungus Nigrospora chinensis, which was named "Nigrospora chinensis victorivirus 1" (NcVV1). The NcVV1 genome is 5283 bp in length, containing two continuous open reading frames (ORFs), ORF1 and ORF2. ORF1 and ORF2 were predicted to encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), respectively. The stop codon of ORF1 overlaps with the start codon of ORF2 by the tetranucleotide sequence AUGA. Phylogenetic analysis based on amino acid sequences of RdRp and CP indicated that NcVV1 clustered with members of the genus Victorivirus in the family Totiviridae. To our knowledge, this was the first report of a mycovirus infecting N. chinensis.

X-ray crystallographic characterization of the SARS-CoV-2 main protease polyprotein cleavage sites essential for viral processing and maturation.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, produces polyproteins 1a and 1ab that contain, respectively, 11 or 16 non-structural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for viral assembly and maturation. Using C-terminally substituted Mpro chimeras, we have determined X-ray crystallographic structures of Mpro in complex with 10 of its 11 viral cleavage sites, bound at full occupancy intermolecularly in trans, within the active site of either the native enzyme and/or a catalytic mutant (C145A). Capture of both acyl-enzyme intermediate and product-like complex forms of a P2(Leu) substrate in the native active site provides direct comparative characterization of these mechanistic steps as well as further informs the basis for enhanced product release of Mpro's own unique C-terminal P2(Phe) cleavage site to prevent autoinhibition. We characterize the underlying noncovalent interactions governing binding and specificity for this diverse set of substrates, showing remarkable plasticity for subsites beyond the anchoring P1(Gln)-P2(Leu/Val/Phe), representing together a near complete analysis of a multiprocessing viral protease. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for antiviral therapeutic development.