Viral Gene Functions Research Articles

Construction and Characterization of a Full-Length Infectious cDNA Clone of a Strain of Watermelon Mosaic Virus Isolated from Melon.

Watermelon mosaic virus (WMV), a member of the genus Potyvirus, causes serious economic losses in cucurbit crops. For molecular biological studies of viruses, it is necessary to construct an infectious clone that can facilitate gene functional analysis. In this study, we constructed an infectious cDNA clone of WMV genomic RNA by Gibson assembly and evaluated its virulence and symptoms on a variety of host plants. A WMV isolate (WMV-IS-me2) collected from melon in Iksan, Jeonbuk Province, Korea in 2015 caused mosaic symptoms on leaves. Four overlapping PCR fragments of the full-length genome of this isolate and the pJL89 binary vector with overhanging ends of 40 bp were amplified by reverse transcription PCR and joined in a single isothermal reaction. The complete nucleotide sequence of the infectious cDNA clone of WMV was determined and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. Following agro-infiltration, an infectious clone (pWMV-M, KACC95145P) systemically induced mosaic symptoms on watermelon (Citrullus lanatus), zucchini (Cucurbita pepo), melon (Cucumis melo), and Nicotiana benthamiana plants. This infectious clone may be useful for screening cucurbit varieties resistant to WMV, as well as for studying viral gene function.

Generation of an infectious cDNA clone for NADC30-like PRRSV.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly significant infectious disease that poses a substantial threat to the global pig industry. In recent years, the NADC30-like strain has gradually emerged as prevalent in China, causing a profound impact on the country's pig farming industry. Therefore, it is important to conduct an in-depth study on the characteristics and gene functions of the NADC30-like strain. An infectious cDNA clone is an indispensable tool for investigating the functions of viral genes. In this current study, we successfully isolated a NADC30-like strain and constructed its full-length infectious cDNA clone. The utilization of this clone will facilitate our investigation into the viral replication, pathogenesis, and immune response associated with the PRRSV NADC30-like strain.

The bacterial and viral communities associated with onion bacterial bulb rot

Microbes occur in complex communities within plants as endophytes and establish a network of interactions that can influence plant health positively or negatively. Positive interactions may be synergistic and confer abiotic and biotic stress tolerance. The aim of this study was to identify bacteria and viruses present in storage onion bulbs that were symptomatic and asymptomatic for bacterial bulb rot, from crops grown in Georgia and Washington states; and to assess their potential role in the bulbs based on functions of bacterial and viral genes detected. DNA was extracted from nine asymptomatic bulbs and nine bulbs displaying symptoms of bacterial bulb rot and subjected to 16S rRNA amplicon sequencing and Metagenomic Assembled Genome (MAG) analysis. The Illumina platform was used to sequence the hypervariable region (V3-V4) of the 16S rRNA gene. The 16S rRNA amplicon profiling revealed the presence of numerous bacteria including potential onion pathogens in the genera Panteoa and Burkholderia. MAG assembly identified P. agglomerans. B. gladioli and B. cepacia, known bulb rot pathogens, including genes linked to fitness and those involved in the Type II and III secretion systems. Eighty-nine unique viral genomes were identified, of which 67 could be classified taxonomically. The bacterial and viral genomes differed significantly in asymptomatic versus symptomatic bulbs. Viral genomes showed evidence of auxiliary metabolic genes (AMGs), including genes involved in fitness and pathogenicity to bacterial hosts. The bulbs hosted endophytic bacteria and viruses, some of which were potentially beneficial, and others potentially pathogenic to onion or as hosts to bacteriophages.

Metabolites from traditional Chinese botanical drugs with anti-hepatitis B virus activity - a review.

Hepatitis B virus (HBV)-related liver disease poses a major threat to human health worldwide. Although interferon and nucleoside analogues are commonly administered for treating chronic HBV infection, their use is limited by considerable side effects, drug resistance and incapacity for HBV elimination. Hence, novel HBV therapeutics are urgently required. For numerous years, traditional Chinese botanical drugs have been widely used to treat HBV-related diseases. The natural metabolites derived from these traditional drugs exhibit significant anti-HBV effects and serve as potential novel drugs for treating HBV. For overall understanding the therapeutic potential of these metabolites, the anti-HBV effects and mechanisms of action of 107 natural metabolites are summarized in this article. Mechanistically, these natural metabolites exert their anti-HBV effects by influencing the expression and function of host and/or viral genes, which differs from the mechanism of action of nucleoside analogues. Indeed, combining natural metabolites with nucleoside analogues can exert synergistic effects. Accordingly, natural metabolites or their chemically modified derivatives represent potential novel drugs and adjuvants for anti-HBV treatment.

The most exposed regions of SARS-CoV-2 structural proteins are subject to strong positive selection and gene overlap may locally modify this behavior.

The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic that emerged in 2019 has been an unprecedented event in international science, as it has been possible to sequence millions of genomes, tracking their evolution very closely. This has enabled various types of secondary analyses of these genomes, including the measurement of their sequence selection pressure. In this work, we have been able to measure the selective pressure of all the described SARS-CoV-2 genes, even analyzed by sequence regions, and we show how this type of analysis allows us to separate the genes between those subject to positive selection (usually those that code for surface proteins or those exposed to the host immune system) and those subject to negative selection because they require greater conservation of their structure and function. We have also seen that when another gene with an overlapping reading frame appears within a gene sequence, the overlapping sequence between the two genes evolves under a stronger purifying selection than the average of the non-overlapping regions of the main gene. We propose this type of analysis as a useful tool for locating and analyzing all the genes of a viral genome when an adequate number of sequences are available.IMPORTANCEWe have analyzed the selection pressure of all severe acute respiratory syndrome coronavirus 2 genes by means of the nonsynonymous (Ka) to synonymous (Ks) substitution rate. We found that protein-coding genes are exposed to strong positive selection, especially in the regions of interaction with other molecules (host receptor and genome of the virus itself). However, overlapping coding regions are more protected and show negative selection. This suggests that this measure could be used to study viral gene function as well as overlapping genes.

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells.

RNA viruses are often cited as a significant factor affecting the populations of both domestic honey bees and wild pollinators. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail the in vitro transcription of the viral genome RNA in 8-10 kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of the Chinese variant sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a widely used lepidopteran immortal cell line (Sf9) and honey bee pupae. Furthermore, the negative strand of the clone was detected in both Sf9 cells and honey bee pupae, indicating active infection and replication. However, the transfection of Sf9 cells was observed in only a limited proportion (less than 10%) of the cells, and the infection did not appear to spread to adjacent cells or form infective virions. The injection of honey bee pupae with 2500 ng of the IE1-SBV plasmid resulted in high infection rates in Apis cerana pupae but low rates in A. mellifera pupae, although the dosage was comparatively high compared with other studies using in vitro transcribed viral RNA. Our findings suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, albeit requiring additional optimization. However, this method holds substantial potential for facilitating the production of clones with various sequence modifications, enabling the exploration of viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid form may foster collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately offering a comprehensive approach to managing virus diseases in the future.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Visual tracking of viral infection dynamics reveals the synergistic interactions between cucumber mosaic virus and broad bean wilt virus 2

Cucumber mosaic virus (CMV) is one of the most prevalent plant viruses in the world, and causes severe damage to various crops. CMV has been studied as a model RNA virus to better understand viral replication, gene functions, evolution, virion structure, and pathogenicity. However, CMV infection and movement dynamics remain unexplored due to the lack of a stable recombinant virus tagged with a reporter gene. In this study, we generated a CMV infectious cDNA construct tagged with a variant of the flavin-binding LOV photoreceptor (iLOV). The iLOV gene was stably maintained in the CMV genome after more than four weeks of three serial passages between plants. Using the iLOV-tagged recombinant CMV, we visualized CMV infection and movement dynamics in living plants in a time course manner. We also examined whether CMV infection dynamics is influenced by co-infection with broad bean wilt virus 2 (BBWV2). Our results revealed that no spatial interference occurred between CMV and BBWV2. Specifically, BBWV2 facilitated the cell-to-cell movement of CMV in the upper young leaves. In addition, the BBWV2 accumulation level increased after co-infection with CMV.

Interspecies/Intergroup Complementation of Orthotospovirus Replication and Movement through Reverse Genetics Systems.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

3′ UTR is critical for viral RNA accumulation of jasmine virus H

Jasmine virus H (JaVH) is a newly reported viral pathogen of jasmine in China and USA. To study the viral gene function and pathogenic mechanism, a full-length infectious clone of JaVH (pXT-JaVHFJ) was constructed under the control of the cauliflower mosaic virus 35S promoter. pXT-JaVHFJ induced a systemic infection in Nicotiana benthamiana plants by Agro-infiltration, which demonstrated that pXT-JaVHFJ was biologically active. Jasmine showed yellow spots after rubbing with total RNA extracted from Agro-infiltrated N. benthamiana, indicating that JaVH was highly associated with yellow mosaic symptoms observed on jasmine. To investigate the occurrence and mutations of the virus, jasmine samples were collected from eight provinces of China and were tested for JaVH. The samples that were tested positive for JaVH were used to determine the complete genome sequences. They were comprised of 3867 or 3868 nucleotides and their genome organizations resembled that we previous reported for JaVH-FJ. Phylogenetic analyses and sequence comparisons suggest that the eight virus isolates were close isolates of JaVH-FJ and the isolate from Jilin Province was most closely related to JaVH-FJ with 99.2% nucleotide identity over the entire genome and 99.7% identity of coat protein. Further comparative analyses of JaVH-FJ and JaVH-JL revealed additional nucleotide differences in the 3′-untranslated region (3′ UTR). An infectious clone of JaVH-JL and chimeric mutants containing JaVH-FJ or JaVH-JL 3′ UTRs were then constructed for further study. The differential accumulation of JaVH with distinct 3′ UTR suggested that the 3′ UTR of JaVH plays a crucial role in viral RNA accumulation.

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data.

The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells.

ABSTRACTEssential genus-specific genes have not been discovered for fowl adenovirus (FAdV), which hampers the development of FAdV-based vectors and attenuated FAdV vaccines. Reverse genetics approaches were employed to construct FAdV-4 mutants carrying deletions or frameshift mutations covering the whole left and right ends of the viral genome. The results of virus rescue and plaque forming experiments illustrated that all the 22 designated ORFs (open reading frames) were dispensable for the replication of FAdV-4 in chicken hepatoma Leghorn male hepatoma (LMH) cells and primary embryo hepatocytes. RNA-seq data demonstrated that ORF28 and ORF29 were not protein-encoding genes, and suggested a promoter (RP1) and an intron in these regions, respectively. The promoter activity of RP1 was further confirmed by reporter gene expression experiments. GAM-1-deleted FAdV-4 formed small plaques, while deletion of GAM-1 together with ORF22 resulted in even smaller ones in LMH cells. Simultaneous deletion of ORF28, ORF29, and GAM-1 led to growth defect of FAdV-4. These facts implied that genus-specific genes contributed to and synergistically affected viral replication, although no single one was essential. Notably, replication of FAdV-4 mutants could be different in vitro and in vivo. XGAM1-CX19A, a GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells, did not kill chicken embryos because virus propagation took place at a very low level in vivo. This work laid a solid foundation for FAdV-4 vector construction as well as vaccine development, and would benefit viral gene function study.IMPORTANCE Identification of viral essential genes is important for adenoviral vector construction. Deletion of nonessential genes enlarges cloning capacity, deletion of essential genes makes a replication-defective vector, and expression of essential genes in trans generates a virus packaging cell line. However, the genus-specific essential genes in FAdV have not been identified. We constructed adenoviral plasmid carrying deletions covering all 22 genus-specific ORFs of FAdV-4, and found that all virus mutants could be rescued and amplified in chicken LMH cells except those that had defects in key promoter activity. These genus-specific genes affected virus growth, but no single one was indispensable. Dysfunction of several genus-specific genes at the same time could make FAdV-4 vectors replication-defective. In addition, the growth of FAdV-4 mutants could be different in LMH cells and in chicken embryos, suggesting the possibility of constructing attenuated FAdV-4 vaccines.

More Positive or More Negative? Metagenomic Analysis Reveals Roles of Virome in Human Disease-Related Gut Microbiome.

Viruses are increasingly viewed as vital components of the human gut microbiota, while their roles in health and diseases remain incompletely understood. Here, we first sequenced and analyzed the 37 metagenomic and 18 host metabolomic samples related to irritable bowel syndrome (IBS) and found that some shifted viruses between IBS and controls covaried with shifted bacteria and metabolites. Especially, phages that infect beneficial lactic acid bacteria depleted in IBS covaried with their hosts. We also retrieved public whole-genome metagenomic datasets of another four diseases (type 2 diabetes, Crohn’s disease, colorectal cancer, and liver cirrhosis), totaling 438 samples including IBS, and performed uniform analysis of the gut viruses in diseases. By constructing disease-specific co-occurrence networks, we found viruses actively interacting with bacteria, negatively correlated with possible dysbiosis-related and inflammation-mediating bacteria, increasing the connectivity between bacteria modules, and contributing to the robustness of the networks. Functional enrichment analysis showed that phages interact with bacteria through predation or expressing genes involved in the transporter and secretion system, metabolic enzymes, etc. We further built a viral database to facilitate systematic functional classification and explored the functions of viral genes on interacting with bacteria. Our analyses provided a systematic view of the gut virome in the disease-related microbial community and suggested possible positive roles of viruses concerning gut health.

Using the CRISPR-Cas System to Solve Porcine Viral Infection-related Issues.

Viral infection-associated diseases seriously affect the development of the swine industry and pose a potential threat to the health of humans. Fortunately, the emergence of CRISPR-Cas has inspired scientists' efforts to address these viral-related issues in pigs using this technology. Based on progress in the field to date, this review summarizes the applications of the CRISPR-Cas system in dissecting the functions of swine viral genes and host factors related to their infections, improving the antiviral ability of pigs, inactivating porcine endogenous retrovirus prior to xenotransplantation, and detecting swine viruses. We also discuss the challenges of the practice of porcine genetic modification and the CRISPR-Cas system's prospects as an important tool for basic virology research and a promising strategy for controlling swine viral infection-related diseases.

SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe morbidity and mortality in humans. It is urgent to understand the function of viral genes. However, the function of open reading frame 10 (ORF10), which is uniquely expressed by SARS-CoV-2, remains unclear. In this study, we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes. Then, mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway. Furthermore, overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B. Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10. Consistent with our observations, in the context of SARS-CoV-2 infection, ORF10 inhibited MAVS expression and facilitated viral replication. In brief, our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.

Deconstructing virus condensation.

Viruses have evolved precise mechanisms for using the cellular physiological pathways for their perpetuation. These virus-driven biochemical events must be separated in space and time from those of the host cell. In recent years, granular structures, known for over a century for rabies virus, were shown to host viral gene function and were named using terms such as viroplasms, replication sites, inclusion bodies, or viral factories (VFs). More recently, these VFs were shown to be liquid-like, sharing properties with membrane-less organelles driven by liquid–liquid phase separation (LLPS) in a process widely referred to as biomolecular condensation. Some of the best described examples of these structures come from negative stranded RNA viruses, where micrometer size VFs are formed toward the end of the infectious cycle. We here discuss some basic principles of LLPS in connection with several examples of VFs and propose a view, which integrates viral replication mechanisms with the biochemistry underlying liquid-like organelles. In this view, viral protein and RNA components gradually accumulate up to a critical point during infection where phase separation is triggered. This yields an increase in transcription that leads in turn to increased translation and a consequent growth of initially formed condensates. According to chemical principles behind phase separation, an increase in the concentration of components increases the size of the condensate. A positive feedback cycle would thus generate in which crucial components, in particular nucleoproteins and viral polymerases, reach their highest levels required for genome replication. Progress in understanding viral biomolecular condensation leads to exploration of novel therapeutics. Furthermore, it provides insights into the fundamentals of phase separation in the regulation of cellular gene function given that virus replication and transcription, in particular those requiring host polymerases, are governed by the same biochemical principles.

Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi's Sarcoma-Associated Herpesvirus-Infected Cells.

Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR-Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80–90%, in epithelial and B-cells carrying multiple copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus–host interactions.