Search Results (33)

The open reading frame 103 (p48) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is one of the 38 core baculovirus genes. p48 has been shown to be essential for the production of infectious budded virions (BVs), nuclear egress of nucleocapsids, envelopment of the nucleocapsid, and embedding of occlusion-derived virions (ODVs) into occlusion bodies (OBs). However, the structure–function relationship of P48 remains unclear. In this study, we showed that four conserved cysteines (C127, C130, C138, and C141) in P48 may form a zinc finger motif based on a predicted structure analysis, and we investigated the roles of these cysteines in P48 function. AcMNPV bacmids lacking p48 or containing mutated p48 were generated. Transfection/infection assays showed that C127, C130, C138, and C141 in P48 were crucial for infectious BV production. Electron microscopy analysis further confirmed that these four cysteines played critical roles in the transport of nucleocapsids out of the nucleus for BV production, and in ODV envelopment. These results demonstrate that the conserved cysteines C127, C130, C138, and C141, related to the putative zinc finger motif, are critical for P48 function in baculovirus infection. Full article

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. Full article

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC’s basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC’s high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC’s basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects. Full article

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72–8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust ‘pan-variant’ antigen-based assays for viruses prone to high mutational rates. Full article

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2–220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low. Full article

The Flavivirus genus of the Flaviviridae family of enveloped single-stranded RNA viruses encompasses more than 70 members, many of which cause significant disease in humans and livestock. Packaging and assembly of the flavivirus RNA genome is essential for the formation of virions, which requires intricate coordination of genomic RNA, viral structural, and nonstructural proteins in association with virus-induced, modified endoplasmic reticulum (ER) membrane structures. The capsid (C) protein, a small but versatile RNA-binding protein, and the positive single-stranded RNA genome are at the heart of the elusive flavivirus assembly process. The nucleocapsid core, consisting of the genomic RNA encapsidated by C proteins, buds through the ER membrane, which contains viral glycoproteins prM and E organized as trimeric spikes into the lumen, forming an immature virus. During the maturation process, which involves the low pH-mediated structural rearrangement of prM and E and furin cleavage of prM in the secretory pathway, the spiky immature virus with a partially ordered nucleocapsid core becomes a smooth, mature virus with no discernible nucleocapsid. This review focuses on the mechanisms of genome packaging and assembly by examining the structural and functional aspects of C protein and viral RNA. We review the current lexicon of critical C protein features and evaluate interactions between C and genomic RNA in the context of assembly and throughout the life cycle. Full article

The immune response elicited by the current COVID-19 vaccinations declines with time, especially among the immunocompromised population. Furthermore, the emergence of novel SARS-CoV-2 variants, particularly the Omicron variant, has raised serious concerns about the efficacy of currently available vaccines in protecting the most vulnerable people. Several studies have reported that vaccinated people get breakthrough infections amid COVID-19 cases. So far, five variants of concern (VOCs) have been reported, resulting in successive waves of infection. These variants have shown a variable amount of resistance towards the neutralising antibodies (nAbs) elicited either through natural infection or the vaccination. The spike (S) protein, membrane (M) protein, and envelope (E) protein on the viral surface envelope and the N-nucleocapsid protein in the core of the ribonucleoprotein are the major structural vaccine target proteins against COVID-19. Among these targets, S Protein has been extensively exploited to generate effective vaccines against COVID-19. Hence, amid the emergence of novel variants of SARS-CoV-2, we have discussed their impact on currently available vaccines. We have also discussed the potential roles of S Protein in the development of novel vaccination approaches to contain the negative consequences of the variants’ emergence and acquisition of mutations in the S Protein of SARS-CoV-2. Moreover, the implications of SARS-CoV-2’s structural proteins were also discussed in terms of their variable potential to elicit an effective amount of immune response. Full article

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA. In addition, progeny rcDNA, newly formed in cytoplasmic nucleocapsids, is also delivered to the nucleus in the same cell to form more cccDNA in a process called intracellular cccDNA amplification or recycling. Here, we focus on recent evidence demonstrating differential effects of HBc in affecting cccDNA formation during de novo infection vs. recycling, obtained using HBc mutations and small molecule inhibitors. These results implicate a critical role of HBc in determining HBV trafficking during infection, as well as in nucleocapsid disassembly (uncoating) to release rcDNA, events essential for cccDNA formation. HBc likely functions in these processes via interactions with host factors, which contributes critically to HBV host tropism. A better understanding of the roles of HBc in HBV entry, cccDNA formation, and host species tropism should accelerate ongoing efforts to target HBc and cccDNA for the development of an HBV cure and facilitate the establishment of convenient animal models for both basic research and drug development. Full article

Hepatitis B virus (HBV) core antigen (HBc) is a structural protein that forms the viral nucleocapsid and is involved in various steps of the viral replication cycle, but its role in the pathogenesis of HBV infection is still elusive. In this study, we generated a mouse monoclonal antibody (mAb) against HBc and used it in antibody-based in situ biotinylation analysis in order to identify host proteins that interact with HBc. HBc antigen was produced with a wheat germ cell-free protein synthesis system and used to immunize mice. Among the established hybridoma clones, a single clone (mAb #7) was selected and further characterized for its ability in the antibody-based in situ biotinylation analysis to collect host proteins that are in the vicinity of HBc. Using mass spectrometry, we identified 215 HBc-interacting host proteins, three of which bind HBc most significantly under hypoxic conditions. Our results indicate that mAb #7 can be used to systematically identify host proteins that interact with HBc under pathophysiological conditions, and thus may be useful to explore the molecular pathways involved in HBV-induced cytopathogenesis. Full article

Emaravirus (Order Bunyavirales; Family Fimoviridae) is a genus comprising over 20 emerging plant viruses with a worldwide distribution and economic impact. Emaraviruses infect a variety of host plants and have especially become prevalent in important long-living woody plants. These viruses are enveloped, with a segmented, single-stranded, negative-sense RNA genome and are transmitted by eriophyid mites or mechanical transmission. Emaraviruses have four core genome segments encoding an RNA-dependent RNA polymerase, a glycoprotein precursor, a nucleocapsid protein, and a movement protein. They also have additional genome segments, whose number varies widely. We report here that the proteins encoded by these segments form three main homology groups: a homolog of the sadwavirus Glu2 Pro glutamic protease; a protein involved in pathogenicity, which we named “ABC”; and a protein of unknown function, which we named “P55”. The distribution of these proteins parallels the emaravirus phylogeny and suggests, with other analyses, that emaraviruses should be split into at least two genera. Reliable diagnosis systems are urgently needed to detect emaraviruses, assess their economic and ecological importance, and take appropriate measures to prevent their spread (such as routine testing, hygiene measures, and control of mite vectors). Additional research needs include understanding the function of emaravirus proteins, breeding resistant plants, and clarifying transmission modes. Full article

The viral envelope is essential for virus maturation. Virus-mediated syncytium formations are induced by viral envelope proteins that cause membrane fusion of the infected cells. Polydnaviridae (Polydnavirus) are enveloped viruses with multiple nucleocapsids, and virions mature in symbiotic parasitoid wasp ovaries. However, the mechanism governing the envelope packaging of multiple nucleocapsids remains unclear. In this study, we used transmission electron microscopy to examine the process whereby multiple nucleocapsids of Microplitis bicoloratus bracovirus are packaged into an envelope and observed envelope-fusion-syncytium formation in symbiotic wasp calyx cells during virus maturation. The virus maturation process in calyx cells comprised four stages: pre-virogenic stroma, virogenic stroma, assembly, and fusion. Each virus contained a single envelope with one nucleocapsid in the assembly stage; multiple envelopes then fused to form a viral envelope with multiple nucleocapsids (i.e., the envelope-fusion-syncytium) around the envelope fusion core in the fusion stage. The envelope-fusion-syncytium then stabilized the virions that were released into the lumen of the ovary across the calyx epithelial layer. The phagocytic calyx epithelial cells on the border of the calyx and ovary lumen cleared the majority of non-enveloped nucleocapsids. In contrast, non-phagocytic calyx epithelial cells with microvilli and a cuticular line between the ovary wall and the lumen remained intact in the ovary lumen. These results indicate that envelope-fusion-syncytium formation is important for packaging multiple nucleocapsids in bracovirus maturation. Full article

White spot syndrome virus (WSSV) has been reported to cause severe economic loss in the shrimp industry. With WSSV being a large virus still under investigation, the 3D structure of its assembly remains unclear. The current study was planned to clarify the 3D structures of WSSV infections in the cell nucleus of red swamp crayfish (Procambarus clarkii). The samples from various tissues were prepared on the seventh day post-infection. The serial sections of the intestinal tissue were obtained for electron tomography after the ultrastructural screening. After 3D reconstruction, the WSSV-associated structures were further visualized, and the expressions of viral proteins were confirmed with immuno-gold labeling. While the pairs of sheet-like structures with unknown functions were observed in the nucleus, the immature virions could be recognized by the core units of nucleocapsids on a piece of the envelope. The maturation of the particle could include the elongation of core units and the filling of empty nucleocapsids with electron-dense materials. Our observations may bring to light a possible order of WSSV maturation in the cell nucleus of the crayfish, while more investigations remain necessary to visualize the detailed viral–host interactions. Full article

Hepatitis C virus (HCV) is the primary pathogen responsible for liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been linked to several aspects of HCV pathology, including oncogenesis, immune evasion and stress responses. We and others have previously shown that C expression in various cell lines activates Ca²⁺ signaling and alters Ca²⁺ homeostasis. In this study, we identified two distinct C protein regions that are required for the activation of Ca²⁺/NFAT signaling. In the basic N-terminal domain, which has been implicated in self-association of C, amino acids 1–68 were critical for NFAT activation. Sedimentation analysis of four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain was not required for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein to function. Our results indicate that targeting of HCV C to the ER is necessary but not sufficient for inducing Ca²⁺/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane. Full article

Helicoverpa assulta is a pest that causes severe damage to tobacco, pepper and other cash crops. A local strain of HearNPV-BJ (formerly Helicoverpa assulta nucleopolyhedrovirus (HeasNPV-DJ0031)) was isolated from infected H. assulta larvae in Beijing, which had been regarded as a new kind of baculovirus in previous studies. Describing the biological characteristics of the strain, including its external morphology, internal structure and the pathological characteristics of the infection of various cell lines, can provide references for the identification and function of the virus. HearNPV-BJ virion was defined as a single-nucleocapsid nucleopolyhedrovirus by scanning electron microscopy. QB-Ha-E-5 (H. armigera) and BCIRL-Hz-AM1 (H. zea) cell lines were sensitive to HearNPV-BJ. Undoubtedly modern developed sequencing technology further facilitates the increasing understanding of various strains. The whole genome sequence of the HearNPV-BJ was sequenced and analyzed. The HearNPV-BJ isolate genome was 129, 800 bp nucleotides in length with a G + C content of 38.87% and contained 128 open reading frames (ORFs) encoding predicted proteins of 50 or over 50 amino acids, 67 ORFs in the forward orientation and 61 ORFs in the reverse orientation, respectively. The genome shared 99% sequence identity with Helicoverpa armigera nucleopolyhedrovirus C1 strain (HearNPV-C1), and 103 ORFs had very high homology with published HearNPV sequences. Two bro genes and three hrs were found to be dispersed along the HearNPV-BJ genome. Three of the highest homologs, ORFs with HearNPV, were smaller due to the earlier appearance of the stop codon with unknown functions. P6.9 of HearNPV-BJ, a structural protein, is distinctly different from that of Autographa californica nucleopolyhedrovirus (AcMNPV); its homology with the corresponding gene in HearNPV-C1 was 93.58%. HearNPV-BJ contains 38 core genes identified in other baculoviruses, and phylogenetic analysis indicates HearNPV-BJ belongs to Alphabaculovirus Group II, same as HearNPV-C1. The resulting data provide a better understanding of virion structure, gene function and character of infection. By supplementing the whole-genome sequencing data and Kimura-2 model index, there is more evidence to indicate that HearNPV-BJ may be a variant of Helicoverpa armigera nucleopolyhedrovirus, which also deepens our understanding of the virus species demarcation criteria. Full article

Despite a preventive vaccine being available, more than 250 million people suffer from chronic hepatitis B virus (HBV) infection, a major cause of liver disease and HCC. HBV infects human hepatocytes where it establishes its genome, the cccDNA with chromosomal features. Therapies controlling HBV replication exist; however, they are not sufficient to eradicate HBV cccDNA, the main cause for HBV persistence in patients. Core protein is the building block of HBV nucleocapsid. This viral protein modulates almost every step of the HBV life cycle; hence, it represents an attractive target for the development of new antiviral therapies. Capsid assembly modulators (CAM) bind to core dimers and perturb the proper nucleocapsid assembly. The potent antiviral activity of CAM has been demonstrated in cell-based and in vivo models. Moreover, several CAMs have entered clinical development. The aim of this review is to summarize the mechanism of action (MoA) and the advancements in the clinical development of CAMs and in the characterization of their mod of action. Full article