Journal of General Virology最新文献

Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.

Species A rotaviruses (RVs), which belong to the family Sedoreoviridae and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.

Translation errors, impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal, including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways, the ubiquitin-proteasome system and selective autophagy. In humans, the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain-Barré syndrome). Non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses, catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm, ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here, we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell, although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells, while transient overexpression of UBA1 favours faster replication kinetics, with higher virus titres and protein levels detected. Moreover, UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.

RNA structures that are functionally important are defined as cis-acting RNA elements because their functions cannot be compensated for in trans. The cis-acting RNA elements in the 3' UTR of coronaviruses are important for replication; however, the mechanism linking the cis-acting RNA elements to their replication function remains to be established. In the present study, a comparison of the biological processes of the interactome and the replication efficiency between the 3' UTR cis-acting RNA elements in coronaviruses, including severe acute respiratory syndrome coronavirus 2, suggests that (i) the biological processes, including translation, protein folding and protein stabilization, derived from the analysis of the cis-acting RNA element interactome and (ii) the architecture of the cis-acting RNA elements and their interactomes are highly correlated with coronavirus replication. In addition, alteration of the interactome using the compound 5-benzyloxygramine can cause reduced coronavirus replication, reinforcing the connection between cis-acting RNA elements and replication by interactome. Together, these results link cis-acting RNA elements to the coronavirus replication and establish a model to analyse the cis-acting RNA elements in the replication of RNA viruses by interactome.

Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here, we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors, we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody, with Chinese hamster ovary cells expressing 20-50 mg l^-1 after 5 days when cells were grown to a density of ~1×10⁶ cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo.

Human immunodeficiency virus (HIV) is an exemplar virus, still the most studied and best understood and a model for mechanisms of viral replication, immune evasion and pathogenesis. In this review, we consider the earliest stages of HIV infection from transport of the virion contents through the cytoplasm to integration of the viral genome into host chromatin. We present a holistic model for the virus-host interaction during this pivotal stage of infection. Central to this process is the HIV capsid. The last 10 years have seen a transformation in the way we understand HIV capsid structure and function. We review key discoveries and present our latest thoughts on the capsid as a dynamic regulator of innate immune evasion and chromatin targeting. We also consider the accessory proteins Vpr and Vpx because they are incorporated into particles where they collaborate with capsids to manipulate defensive cellular responses to infection. We argue that effective regulation of capsid uncoating and evasion of innate immunity define pandemic potential and viral pathogenesis, and we review how comparison of different HIV lineages can reveal what makes pandemic lentiviruses special.

Bromoviridae is a family of plant viruses with tripartite, positive-sense RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain sub-genomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and may be transmitted between hosts mechanically, via pollen, or non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomatoes, cucurbits, bananas, fruit trees, common beans and alfalfa. Since the adoption of metagenomic high-throughput sequencing methodologies, there has been a notable increase in the number of species in the genus Ilarvirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bromoviridae, which is available at ictv.global/report/bromoviridae.

The Betacoronavirus murine hepatitis virus (MHV) is an important model system for studying coronavirus (CoV) molecular and cell biology. Despite this, few reagents for MHV are available through repositories such as ATCC or Addgene, potentially limiting the widespread adoption of MHV as a tractable model system. To overcome some challenges inherent in the existing MHV reverse genetics systems, we developed a plasmid-launched transformation-associated recombination (TAR) cloning-based system to assemble the MHV (strain A59; MHV-A59) genome. Following assembly in yeast, virus replication was launched by transfecting the fully assembled genome into HEK-293T cells. MHV-A59 recovered using this TAR cloning-based approach (WT_TAR MHV-A59) replicated with kinetics identical to the virus recovered using a ligation- and T7-based approach (WT_LIG MHV-A59). Additionally, WT_TAR MHV-A59 can be detected at least 10 h post-transfection without requiring additional nucleocapsid (N) provided in trans. Lastly, we demonstrated the tractability of this TAR cloning-based system by recovering MHV-A59 expressing an 11 amino acid-containing HiBiT tag fused to the C-terminus of spike (S). While this virus, S_C MHV-A59, replicated with reduced kinetics compared to WT_TAR MHV-A59, the kinetics of virion production could be measured over time directly from the supernatant. This report represents the first plasmid-launched, TAR cloning-based system for MHV-A59. Furthermore, it describes a new reporter virus that could be used to study early steps during MHV-A59 entry and be used in the screening of antiviral compounds. To support future research with MHV-A59, we have made the necessary plasmids for this system available through ATCC.

Nudiviruses (family Nudiviridae) are double-stranded DNA viruses that infect various insects and crustaceans. Among them, Heliothis zea nudivirus 1 (HzNV-1) represents the rare case of a lepidopteran nudivirus inducing a sexual pathology. Studies about molecular pathological dynamics of HzNV-1 or other nudiviruses are scarce. Hence, this study aims to provide a transcriptomic profile of HzNV-1 in an ovary-derived cell line of Helicoverpa zea (HZ-AM1), during early (3, 6 and 9 h post-infection) and advanced (12 and 24 h post-infection) stages of infection. Total RNA was extracted from both virus- and mock-infected cells, and RNA-seq analysis was performed to examine both virus and host transcriptional dynamics. Hierarchical clustering was used to categorize viral genes, while differential gene expression analysis was utilized to pinpoint host genes that are significantly affected by the infection. Hierarchical clustering classified the 154 HzNV-1 genes into four temporal phases, with early phases mainly involving transcription and replication genes and later phases including genes for virion assembly. In addition, a novel viral promoter motif was identified in the upstream region of early-expressed genes. Host gene analysis revealed significant upregulation of heat shock protein genes and downregulation of histone genes. The identification of temporal patterns in viral gene expression enhances the molecular understanding of nudivirus pathology, while the identified differentially expressed host genes highlight the key pathways most hijacked by HzNV-1 infection.