MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance.

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic ß cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

SPC25 promotes hepatocellular carcinoma metastasis via activating the FAK/PI3K/AKT signaling pathway through ITGB4.

Hepatocellular carcinoma (HCC) is a malignant tumor with a high metastatic rate. Recent studies have shown that the mitosis­associated spindle­assembly checkpoint regulatory protein spindle pole body component 25 homolog (SPC25) promotes HCC progression, although the underlying mechanism has yet to be fully elucidated. The aim of the present study was to investigate the mechanism through which SPC25 may promote HCC progression in greater detail. First, the expression of SPC25 was analyzed in publicly available databases to explore the association between SPC25 and HCC metastasis. Western blotting was subsequently performed to examine the level of SPC25 expression in different HCC cell lines. SPC25 was then silenced in HCCLM3 and Huh7 cells, and the effects of SPC25 silencing were investigated using cell proliferation, wound­healing, Transwell migration assays and an in vivo mouse model. Finally, the mechanism of SPC25 action with respect to the promotion of HCC metastasis was explored using microarray analysis and rescue experiments. The results obtained demonstrated that SPC25 is highly expressed in HCC, and this high level of expression is associated with poor prognosis and metastasis. Moreover, SPC25 silencing led to a marked inhibition of the invasion and migration of HCC cells both in vitro and in vivo. The gene­expression profiling and mechanistic experiments suggest that SPC25 preferentially influences the expression of genes associated with extracellular matrix (ECM)­integrin interactions, including integrin subunit ß4 (ITGB4), an upstream element of the integrin pathway. ITGB4 upregulation partly reversed the decline in cell invasion and migration capacities that resulted from SPC25 silencing. Furthermore, deleting both SPC25 and ITGB4 caused a decrease in the phosphorylation of focal adhesion kinase (FAK), phosphoinositide 3­kinase (PI3K) and AKT, which are downstream elements of the integrin pathway. Taken together, the results of the present study demonstrated the important role of SPC25 as a prognostic indicator and as a promoter of metastasis in HCC, and the underlying mechanism of its action has been partially elucidated, suggesting that SPC25 could be used as a biomarker and as a target for therapeutic intervention in the treatment of HCC.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System.

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which consists of viruses with a genome of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (PNTD) coordinates the C-terminal accessory domains to produce a "compacted" L conformation. Despite this advancement, the role of the three phosphorylation sites in PNTD has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between PNTD and the L protein C-terminal domain (LCTD), we demonstrated our ability to sensitively test for changes in the interface between the two proteins. This method showed that the binding site for PNTD on LCTD is longer than was previously appreciated. We demonstrated that phosphorylation of PNTD modulates its interaction with LCTD and used a minigenome reporter system to validate the functional significance of the PNTD-LCTD interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated PNTD displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of PNTD modulates its cofactor and conformational regulatory activities with L.IMPORTANCE Polymerase-cofactor interactions like those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Despite cofactor phosphorylation being present in most of these interactions, what effect if any it has on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. As phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggest that the structural picture of L during infection might be more complex than previously appreciated.

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

The Connector Domain of Vesicular Stomatitis Virus Large Protein Interacts with the Viral Phosphoprotein.

Vesicular stomatitis virus (VSV) is an archetypical member of Mononegavirales, viruses with a genome of negative-sense single-stranded RNA (-ssRNA). Like other viruses of this order, VSV encodes a unique polymerase, a complex of viral L (large, the enzymatic component) protein and P (phosphoprotein, a cofactor component). The L protein has a modular layout consisting of a ring-shaped core trailed by three accessory domains and requires an N-terminal segment of P (P N-terminal disordered [PNTD]) to perform polymerase activity. To date, a binding site for P on L had not been described. In this report, we show that the connector domain of the L protein, which previously had no assigned function, binds a component of PNTD We further show that this interaction is a positive regulator of viral RNA synthesis, and that the interfaces mediating it are conserved in other members of Mononegavirales Finally, we show that the connector-P interaction fits well into the existing structural information of VSV L.IMPORTANCE This study represents the first functional assignment of the connector domain of a Mononegavirales L protein. Furthermore, this study localizes P polymerase cofactor activity to specific amino acids. The functional necessity of this interaction, combined with the uniqueness of L and P proteins to the order Mononegavirales, makes disruption of the P-connector site a potential target for developing antivirals against other negative-strand RNA viruses. Furthermore, the connector domain as an acceptor site for the P protein represents a new understanding of Mononegavirales L protein biology.

Transcriptome analysis of differential gene expression in the longissimus dorsi muscle from Debao and landrace pigs based on RNA-sequencing.

RNA-seq analysis was used to identify differentially expressed genes (DEGs) at the genetic level in the longissimus dorsi muscle from two pigs to investigate the genetic mechanisms underlying the difference in meat quality between Debao pigs and Landrace pigs. Then, these DEGs underwent functional annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) analyses. Finally, the expression levels of specific DEGs were assessed using qRT-PCR. The reference genome showed gene dosage detection of all samples which showed that the total reference genome comprised 22342 coding genes, including 14743 known and 190 unknown genes. For detection of the Debao pig genome, we obtained 14168 genes, including 13994 known and 174 unknown genes. For detection of the Landrace pig genome, we obtained 14404 genes, including 14223 known and 181 unknown genes. GO analysis and KEGG signaling pathway analysis show that DEGs are significantly related to metabolic regulation, amino acid metabolism, muscular tissue, muscle structure development etc. We identified key genes in these processes, such as FOS, EGR2, and IL6, by PPI network analysis. qRT-PCR confirmed the differential expression of six selected DEGs in both pig breeds. In conclusion, the present study revealed key genes and related signaling pathways that influence the difference in pork quality between these breeds and could provide a theoretical basis for improving pork quality in future genetic thremmatology.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

Distribution and molecular diversity of three cucurbit-infecting poleroviruses in China.

Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0-79.2% nucleotide sequence identity with MABYV isolates, and 69.7-70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3-76.5%. In contrast, the nucleotide identities within each species were 93.2-98.7% (CABYV), 98.1-99.9% (MABYV), and 96.1-98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.

Complete sequence analysis reveals two distinct poleroviruses infecting cucurbits in China.

The complete RNA genomes of a Chinese isolate of cucurbit aphid-borne yellows virus (CABYV-CHN) and a new polerovirus tentatively referred to as melon aphid-borne yellows virus (MABYV) were determined. The entire genome of CABYV-CHN shared 89.0% nucleotide sequence identity with the French CABYV isolate. In contrast, nucleotide sequence identities between MABYV and CABYV and other poleroviruses were in the range of 50.7-74.2%, with amino acid sequence identities ranging from 24.8 to 82.9% for individual gene products. We propose that CABYV-CHN is a strain of CABYV and that MABYV is a member of a tentative distinct species within the genus Polerovirus.