Attenuated Viral Replication of Avian Infectious Bronchitis Virus with a Novel 82-Nucleotide Deletion in the 5a Gene Indicates a Critical Role for 5a in Virus-Host Interactions.

ABSTRACT

We previously found that a deletion in γ-coronavirus Infectious bronchitis virus (IBV) accessory gene 5a is critical for decreased viral pathogenicity in chickens. Here, we systematically analyzed IBV virus infection invasion, genome replication, subgenomic mRNA (sgmRNA) synthesis, protein synthesis, and virion release. The ability of the mutant IBV strain rYN-Δ5a to invade susceptible cells was not significantly different from that of parental rYN. However, compared with rYN, the level of sgmRNA synthesis and genome replication after cell entry by rYN-Δ5a was significantly lower in the early stage, resulting in a significantly lower level of nucleoprotein (N) synthesis and a consequent significantly lower number of offspring viruses released into the supernatant. The detected 5a protein was diffusely distributed in the cytoplasm and perinuclear area. We identified 16 differentially expressed host proteins, 8 of which were found to be host nuclear and cytoplasmic transport-related proteins. Coimmunoprecipitation revealed an interaction between hemagglutinin (HA)-tagged TNPO1, TNPO3, XPO1, XPOT, RanBP1, and EIF2B4 proteins and Flag-tagged 5a protein, and laser confocal microscopy confirmed 5a protein colocalization with these proteins, indicating that 5a protein can cause changes in the host protein localization. These host proteins promote the nuclear localization of N proteins, so we believe that 5a protein can hijack host nucleoplasmic transport-related proteins to help N enter the nucleus. This may involve regulating the cell cycle to promote the optimal intracellular conditions for virus assembly or by participating in the regulation of nucleolar function as a strategy to optimize virus replication. IMPORTANCE Coronaviruses (CoVs) have a huge impact on humans and animals. It is important for the prevention and control of the viruses to assess the molecular mechanisms related to virulence attenuation. Here, we systematically analyzed a single cycle of virus infection by γ-CoV IBV lacking accessory protein 5a. We observed that a 5a deletion in the IBV genome affected virus replication and sgmRNA synthesis early in the virus life cycle, leading to decreases in protein synthesis, offspring virus assembly, and virion release in chicken embryonic kidney cells. IBV 5a protein was found to interact with multiple host nuclear and cytoplasmic transport- and translation-related proteins, which can also interact with IBV N and relocate it into the cell nucleus. These findings provide a comprehensive view regarding the importance of IBV accessory protein 5a and an important theoretical basis for studying the interaction between coronavirus and host cell proteins.