Interplay of the ubiquitin proteasome system and the innate immune response is essential for the replication of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNß) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNß. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.

The N-terminal residual arginine19 of influenza A virus NS1 protein is required for its nuclear localization and RNA binding.

RNA binding ability and cellular distribution are important for nonstructural protein 1 (NS1) of influenza A virus to act as a viral regulatory factor to control virus life cycle. In this study, we identified that the N-terminal residues 19-21 of NS1 are a highly conserved motif depending on all the available NS1 full length sequence of H5N1 influenza A virus from NCBI database. Site-directed mutation analysis demonstrated that the R19 residue of NS1 is critical for its RNA binding and nuclear localization. Furthermore, the residue R19 of NS1 was identified to be critical for regulating M1 mRNA splicing and NS1 nuclear export. Biological analysis of the rescued viruses indicated that the R19A mutation of NS1 did not interfere the replication of H5N1 virus during infection and attenuated the virulence of H5N1 virus in mice.

Construction of an infectious bronchitis virus vaccine strain carrying chimeric S1 gene of a virulent isolate and its pathogenicity analysis.

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.

Protein Interactions Network of Porcine Circovirus Type 2 Capsid With Host Proteins.

Virus-host interaction is a tug of war between pathogenesis and immunity, followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation. Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies. To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein, we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry (LC-MS) approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney (PK-15) cells. Further, a protein-protein interactions (PPIs) network was plotted, and the PCV2 Cap-interacting host proteins were potentially involved in protein binding, DNA transcription, metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment. Verification in vitro assay demonstrated that eight cellular proteins, namely heterogeneous nuclear ribonucleoprotein C, nucleophosmin-1, DEAD-box RNA helicase 21, importin ß3, eukaryotic translation initiation factor 4A2, snail family transcriptional repressor 2, MX dynamin like GTPase 2, and intermediate chain 1 interacted with PCV2 Cap. Thus, this work effectively provides useful protein-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and pathogenesis.

Varicella-Zoster Virus Expresses Multiple Small Noncoding RNAs.

Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells.IMPORTANCE Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts that is likely to reduce viral replication. Since sncRNAs have been suggested to be potential targets for antiviral therapies, identification of these molecules in VZV may provide a new direction for development of treatments for painful herpes zoster.

Hepatitis E virus ORF1 encoded macro domain protein interacts with light chain subunit of human ferritin and inhibits its secretion.

Hepatitis E Virus (HEV) is the major causative agent of acute hepatitis in developing countries. Its genome has three open reading frames (ORFs)-called as ORF1, ORF2, and ORF3. ORF1 encodes nonstructural polyprotein having multiple domains, namely: Methyltransferase, Y domain, Protease, Macro domain, Helicase, and RNA-dependent RNA polymerase. In the present study, we show that HEV-macro domain specifically interacts with light chain subunit of human ferritin (FTL). In cultured hepatoma cells, HEV-macro domain reduces secretion of ferritin without causing any change in the expression levels of FTL. This inhibitory effect was further enhanced upon Brefeldin-A treatment. The levels of transferrin Receptor 1 or ferroportin, two important proteins in iron metabolism, remained unchanged in HEV-macro domain expressing cells. Similarly, there were no alterations in the levels of cellular labile iron pool and reactive oxygen species, indicating that HEV-macro domain does not influence cellular iron homeostasis/metabolism. As ferritin is an acute-phase protein, secreted in higher level in infected persons and HEV-macro domain has the property of reducing synthesis of inflammatory cytokines, we propose that by directly binding to FTL, macro domain prevents ferritin from entering into circulation and helps in further attenuation of the host immune response.

Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment.