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Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO 4 and R43-PO 4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. [ABSTRACT FROM AUTHOR]

Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) non-structural protein 2 (NS2) encodes an autoprotease activity that is essential for virus replication and thus represents an attractive anti-viral target. Recently, we demonstrated that a series of epoxide-based compounds, previously identified as potent inhibitors of the clotting factor, FXIII, also inhibited NS2-mediated proteolysis in vitro and possessed anti-viral activity in cell culture models. This suggested that a selective small molecule inhibitor of the NS2 autoprotease represents a viable prospect. In this independent study, we applied a structure-guided virtual high-throughput screening approach in order to identify a lead-like small molecule inhibitor of the NS2 autoprotease. This screen identified a molecule that was able to inhibit both NS2-mediated proteolysis in vitro and NS2-dependent genome replication in a cell-based assay. A subsequent preliminary structure–activity relationship (SAR) analysis shed light on the nature of the active pharmacophore in this compound and may inform further development into a more potent inhibitor of NS2 mediated proteolysis. [ABSTRACT FROM AUTHOR]

Background: Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed. Methods and Results: Using site-directed mutagenesis at the NS1-2A cleavage site of Murray Valley encephalitis virus (MVEV), we confirmed the dominant role of conserved octapeptide residues for efficient NS1-2A cleavage, while changes at variable and the P1' residues were mostly tolerated. However, digressions from the consensus cleavage motif derived from studies on DENV were also found. Thus, comparison of the impact on cleavage of mutations at the NS1-2A junction of MVEV and DENV showed virus-specific differences at both conserved and variable residues. Conclusion: We show, with subgenomic expression and infectious clone-derived mutants of MVEV that conserved residues in the flavivirus octapeptide motif can be replaced with a different amino acid without markedly reducing cleavage efficiency of NS1 and NS2A. [ABSTRACT FROM AUTHOR]

To explore the expected role of Bovine Viral Diarrhea Virus (BVDV) in pneumonia in cattle, cattle lungs (n=242) showing signs of pneumonia were collected from slaughter houses of three different localities located at Northern, Central and Western Sudan during 2010-2013. The collected samples were tested for the presence of BVDV antigen using Enzyme-Linked Immunosorbent Assay (ELISA), and Fluorescent Antibody Test (FAT). Twenty six (10.7%) out of 242 samples were found to be positive for BVDV. Positive results were seen in all the three studied areas, with the highest prevalence (16.7%; n=4/24) at Gezira State in Central Sudan. BVDV genome could be detected in all ELISA positive samples. The results indicated the existence of BVDV infection in cattle in different areas in Sudan, and its possible association with respiratory infections in cattle. Analysis using BLAST indicated that the sequence was identical to the previously reported BVDV-1 (GenBank accession AF220247.1.); nucleotide A was found in our study at position 9 of our sequence, whereas T was present instead in the reference virus. This is the first report of detecting BVDV antigen, genome, and its sequence analysis collected from cattle lungs in Sudan. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is one of the most important viral encephalitis, caused by the Japanese encephalitis virus (JEV). The function of non-structural protein 2B (NS2B) mostly remains unclear. In our study, NS2B of Japanese encephalitis virus (JEV) was expressed in Escherichia coli and purified by dialysis. After fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from NS2B protein immunized mice, three clones of monoclonal antibodies (MAbs), named 1B9, 3E12, and 4E6, were generated. The specificity and sensitivity of MAbs were demonstrated by ELISA, indirect immunofluorescence assay, and Western blot. These MAbs will be useful in further exploration of the functions of NS2B and the pathogenesis of Japanese encephalitis virus. [ABSTRACT FROM AUTHOR]

Human coronavirus HKU1 (HCoV-HKU1), which mainly causes acute self-limited respiratory-tract infections, belongs to group A of the Betacoronavirus genus. Coronavirus genomes encode 16 nonstructural proteins (nsp1-16), which assemble into a large replication-transcription complex mediating virus propagation. Nonstructural protein 9, which binds to the single-stranded DNA/RNA, has been shown to be indispensible for viral replication. Interestingly, a functional mutant (N60K) of nsp9 was identified to compensate for a 6 nt insertion mutation of the 3′-untranslated region (UTR), which is critical for viral RNA synthesis. It has been proposed that the N60K mutation may cause certain conformational changes of nsp9 to rescue the defective insertion mutant. To further investigate the underlying structural mechanism, the N60K mutant of nsp9 from HCoV-HKU1 was successfully crystallized in this study. The crystals diffracted to 2.6 Å resolution and belonged to space group P212121, with unit-cell parameters a = 31.9, b = 85.0, c = 95.0 Å. Two molecules were identified per asymmetric unit. [ABSTRACT FROM AUTHOR]

Highlights: [•] Dengue virus NS2B–NS3 protease assumes closed conformation in complex with BPTI. [•] Solution conformation is different from crystal structure. [•] There is no evidence for significant population of open conformations in solution. [•] The closed conformation is the best template for rational drug discovery. [ABSTRACT FROM AUTHOR]