Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Ultrastructural characterization of membranous torovirus replication factories.

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.

Enteric infections with coronaviruses and toroviruses.

Many enteric viruses are difficult or impossible to propagate in tissue culture. Coronaviruses and toroviruses are large, enveloped, plus-strand RNA viruses in the order Nidovirales that cause enteric disease in young pigs, cows, dogs, mice, cats and horses. Two different serogroups of mammalian coronaviruses cause frequent respiratory infections in humans, and coronaviruses and toroviruses have been implicated in human diarrhoeal disease by immunoelectron microscopy. However, there is as yet no consensus about the importance of these enveloped viruses in human diarrhoea, and little is known about their genetic variability. The large spike (S) glycoprotein is an important determinant of species specificity, tissue tropism and virulence of coronavirus infection. To infect enterocytes, both S glycoproteins and the viral envelope must resist degradation by proteases, low and high pH, and bile salts. One specific site on the S glycoprotein of bovine coronavirus must be cleaved by an intracellular protease or trypsin to activate viral infectivity and cell fusion. S glycoprotein binds to specific receptors on the apical membranes of enterocytes, and can undergo a temperature-dependent, receptor-mediated conformational change that leads to fusion of the viral envelope with host membranes to initiate infection. Analysing spike-receptor interactions may lead to new ways to propagate these enteric viruses as well as new strategies for development of novel antiviral drugs.