E1B-55K Is a Phosphorylation-Dependent Transcriptional and Posttranscriptional Regulator of Viral Gene Expression in Human Adenovirus C5 Infection.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

Cold Exposure-Induced Up-Regulation of Hsp70 Positively Regulates PEDV mRNA Synthesis and Protein Expression In Vitro.

Porcine epidemic diarrhea (PED) is a highly contagious, intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV as an emerging and re-emerging epizootic virus of swine causes substantial economic losses to the pig industry in China and other countries. In China, the occurrence of PED shows significant seasonal variations, usually outbreak during the winter season. The epidemic characteristics of PED may be highly correlated with the changes of ambient temperature. However, molecular mechanism on the seasonal occurrence of PED still remains unclear. It has been widely observed that low ambient temperature up-regulates the expression of host heat shock protein 70 (Hsp70). Here, we showed that nucleotide and protein levels of Hsp70 were up-regulated in the intestinal of cold exposed pig and cold exposed Vero E6 cells. We found that overexpression of Hsp70 could increase PEDV mRNA synthesis and protein expression in Vero E6 and IPEC-J2 cells, while the siRNAs mediated knockdown of Hsp70 and VER155008 mediated inhibition of Hsp70 resulted in inhibition of viral mRNA synthesis and protein expression in Vero E6 cells. These data suggested that Hsp70 positively regulated PEDV mRNA synthesis and protein expression, which being helpful for understanding the seasonality of PED epidemics and development of novel antiviral therapies in the future.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.