The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.The single-stranded RNA genomes of the Mononegavirales range from ϳ11,000 to 18,000 nucleotides in length and contain a series of tandemly linked genes separated by nontranscribed sequences (32, 34). The viral RNA-dependent RNA polymerase (vRNAP) that transcribes the nucleocapsid protein (NP)-encapsidated RNA into 5Ј capped and 3Ј polyadenylated mRNAs minimally consists of two proteins, the phosphoprotein (P) and the large polymerase protein (L) (22). The viral RNA polymerase is thought to bind the genomic RNA at a single 3Ј entry site and to transcribe the genome by a sequential and polar process. The same vRNAP also replicates viral RNA genome (1,2,21,28). Regulation of the switch between viral RNA transcription and replication is not well understood. It is thought that the phosphorylation status of the P protein may be critical in regulating RNA synthesis (15, 52). For vesicular stomatitis virus (VSV), mutations altering the phosphorylation of the P protein that differentially affect RNA transcription or RNA genome replication have been identified (15,16). Similarly, mutations in the P protein of respiratory syncytial virus (RSV) that affect viral RNA synthesis have been identified (5,20,38). While host kinases are thought to phosphorylate nonsegmented negative-stranded RNA virus (NNSV) P proteins, little is known about the identity of the specific host kinases required. For all paramyxoviruses examined, full activity of RNA synthesis by purified P-L complex with NP-encapsidated RNA ...
IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-β. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5′-cap and 3′-poly (A), can activate IFN expression through an RNase L-MDA5 pathway. RIG-I | RNA polymerase IFNs play a critical role in innate immune responses to viral infections. Viruses trigger expression of IFN-β in infected cells, and IFN-β can lead to activation of IFN-α expression through phosphorylation of IFN regulatory factor 7 (1, 2). IFNs induce an antiviral state in cells that inhibits the spread of infection. MDA5 (melanoma differentiation-associated gene 5), an RNA helicase, plays an essential role in the activation of IFN expression (3). MDA5 is involved in the cytoplasmic sensing of infections by some RNA viruses (4). Recognition of RNA molecules generated during viral infections by MDA5 leads to activation of IFN-β promoter stimulator (IPS)-1, NF-κB, and IFN expression (5). How MDA5 differentiates between self and nonself RNA is unclear. It has been reported that stable, long, double-stranded (ds) RNA structures greater than 2 kb in size, presumably with 5′-triphosphates, generated during RNA virus infection (not typical of self RNA) may serve as a distinguishing factor for MDA5-specific recognition (6). Long, synthetic dsRNA polymers of poly(I):poly(C) are often used as a surrogate for the putative activator of MDA5 (7). A natural single-stranded (ss) RNA trigger for MDA5 has not been identified.The role of MDA5 in regulating IFN expression was first reported in studies of parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (3, 8). PIV5 is a prototypical paramyxovirus in a family of nonsegmented, negative-stranded RNA viruses that includes many important human and animal pathogens, including mumps virus, measles virus, Nipah virus, and respiratory syncytial virus (9). The viral RNA-dependent RNA polymerase, minimally consisting of the L protein and the P protein, transcribes the nucleocapsid protein (NP or N)-encapsidated viral genome RNA into 5′ capped and 3′ polyadenylated mRNAs (10). The V protein of PIV5, a component of PIV5 virions (∼350 molecules per virion) is a multifunctional protein with important roles in viral pathogenesis. The V protein Cterminal domain contains seven cysteine residues, resembling a zinc finger...
Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005–2006 with over reported 5,000 cases which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004–2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuVΔSH). rMuVΔSH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuVΔSH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection.
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