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Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

ABSTRACT: Avian metapneumovirus (aMPV) is one of the major causes of serious respiratory infections of poultry and leads to considerable economic losses to food animal production worldwide. Here, we show that double stranded short interfering RNA (siRNA) molecules corresponding to aMPV phosphoprotein (P) gene silence P RNA and protein expression. These siRNAs broadly reduced the expression of other viral proteins in addition to P, but did not have a discernable effect on cellular protein expression. The exposure of cells to P-specific siRNAs also led to inhibition of virus replication as evidenced by marked reduction in the progeny virion titers. Taken together, the findings suggest that exogenous P silencing siRNAs can inhibit aMPV replication with potential implications in the design of novel siRNA based prophylactics.

SUBMITTER: Munir S

PROVIDER: S-EPMC7114220 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

Munir Shirin S Kaur Kuljeet K Kapur Vivek V

Antiviral research 20051115 1

Avian metapneumovirus (aMPV) is one of the major causes of serious respiratory infections of poultry and leads to considerable economic losses to food animal production worldwide. Here, we show that double stranded short interfering RNA (siRNA) molecules corresponding to aMPV phosphoprotein (P) gene silence P RNA and protein expression. These siRNAs broadly reduced the expression of other viral proteins in addition to P, but did not have a discernable effect on cellular protein expression. The e ...[more]

PMID: 16310868

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Project description:UnlabelledHuman metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivoImportanceThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis in vivo Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.

Project description:Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

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Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

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Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

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