Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

BPR2-D2 targeting viral ribonucleoprotein complex-associated function inhibits oseltamivir-resistant influenza viruses.

OBJECTIVES: The emergence of oseltamivir-resistant viruses raised the global threat with regard to influenza virus infection. To develop alternative antiviral agents against influenza virus infection is significant and urgent. METHODS: A neutralization test was applied as a screening assay and a plaque reduction assay was used for confirmation. Expression plasmids for viral ribonucleoproteins (RNPs) and a plasmid that allowed expression of a pseudoviral reporter RNA were transfected into cells to investigate the effects of a novel antiviral compound on viral RNA synthesis. RESULTS: BPR2-D2 was identified as a novel inhibitor against influenza virus from a hit obtained from high throughput screening of 20 000 or more compounds. BPR2-D2 exhibited an excellent antiviral efficacy for the oseltamivir-resistant virus (EC(50) ranging from 0.021 to 0.040 microM). No resistant virus was produced throughout 20 passages in the presence of BPR2-D2, whereas oseltamivir-resistant virus was generated at passage 8 using the same experimental system. A molecular target other than neuraminidase (NA) was found because BPR2-D2 inhibited the synthesis of viral RNA that was driven by influenza viral RNP in a transfection assay. BPR2-D2 also exhibited a broad antiviral spectrum against various strains of influenza A and influenza B viruses. CONCLUSIONS: BPR2-D2 was identified as a novel inhibitor of influenza virus. It may target viral RNPs that are responsible for viral RNA synthesis. Targeting different molecules compared with NA allows BPR2-D2 to inhibit oseltamivir-resistant viruses.

Mutations at alternative 5' splice sites of M1 mRNA negatively affect influenza A virus viability and growth rate.

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.

HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids.

Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature.

Serotype-specific detection of enterovirus 71 in clinical specimens by DNA microchip array.

Enterovirus 71 is an important pathogen that causes high morbidity and mortality in children in Taiwan. Virus isolation in cell cultures has been the standard method for enterovirus 71 identification in Clinical Virology Laboratories. However, virus isolation takes 5-10 days when using cell culture. A microchip for enterovirus 71 detection was developed as an alternative diagnostic method. The novel approach is based on hybridization of amplified DNA specimens with oligonucleotide DNA probes immobilized on a microchip. Two oligonucleotides were used as detection probes, the pan-enterovirus sequence located in the 5'-noncoding region (5'-NCR) and the enterovirus 71-specific sequence located in the VP2 region. The diagnostic procedure takes 6 h. One hundred specimens identified as enteroviruses by viral cultures were tested using this microchip, including 67 enterovirus 71 specimens. The sensitivity of the novel method is 89.6% and its specificity is 90.9%. The enterovirus 71-microchip can detect the amplicon derived from viral RNA corresponding to 1-10 virions in a clinical specimen. Microchip array is a potential diagnostic method for identification of enterovirus in the future.

Genomic signatures of human versus avian influenza A viruses.

Position-specific entropy profiles created from scanning 306 human and 95 avian influenza A viral genomes showed that 228 of 4591 amino acid residues yielded significant differences between these 2 viruses. We subsequently used 15,785 protein sequences from the National Center for Biotechnology Information (NCBI) to assess the robustness of these signatures and obtained 52 "species-associated" positions. Specific mutations on those points may enable an avian influenza virus to become a human virus. Many of these signatures are found in NP, PA, and PB2 genes (viral ribonucleoproteins [RNPs]) and are mostly located in the functional domains related to RNP-RNP interactions that are important for viral replication. Upon inspecting 21 human-isolated avian influenza viral genomes from NCBI, we found 19 that exhibited > or =1 species-associated residue changes; 7 of them contained > or =2 substitutions. Histograms based on pairwise sequence comparison showed that NP disjointed most between human and avian influenza viruses, followed by PA and PB2.

Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities.

The 3C protease (3C(pro)) of enterovirus 71 (EV71) is a good molecular target for drug discovery. Notably, this protease was found to possess RNA-binding activity. The regions responsible for RNA binding were classified as 'KFRDI' (positions 82-86) and 'VGK' (positions 154-156) in 3C(pro) by mutagenesis study. Although the RNA-binding regions are structurally distinct from the catalytic site of EV71 3C(pro), mutations in the RNA-binding regions influenced 3C(pro) proteolytic activity. In contrast, mutations at the catalytic site had almost no influence on RNA binding ability. We identified certain mutations within 3C(pro) which abrogated both the RNA-binding activity of the expressed, recombinant, protease and the ability to rescue virus from an infectious full-length clone of EV71 (pEV71). Interestingly, mutation at position 84 from Arg(R) to Lys(K) was found to retain good RNA binding and proteolytic activity for the recombinant 3C(pro); however, no virus could be rescued when pEV71 with the R84K mutation was introduced into the infectious copy. Together, these results may provide useful information for using 3C(pro) as the molecular target to develop anti-EV71 agents.