Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

PABPC4 Broadly Inhibits Coronavirus Replication by Degrading Nucleocapsid Protein through Selective Autophagy.

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.

The autoimmune response elicited by mouse hepatitis virus (MHV-A59) infection is modulated by liver tryptophan-2,3-dioxygenase (TDO).

In a previous work we demonstrated that inhibition of mouse indoleamine 2,3-dioxygenase (IDO) by methyltryptophan (MT) exacerbated the pathological actions of mouse hepatitis virus (MHV-A59) infection, suggesting that tryptophan (TRP) catabolism was involved in viral effects. Since there is a second enzyme that dioxygenates TRP, tryptophan-2, 3-dioxygenase (TDO), which is mainly located in liver, we decided to study its role in our model of MHV-infection. Results showed that in vivo TDO inhibition by LM10, a derivative of 3-(2-(pyridyl) ethenyl) indole, resulted in a decrease of anti- MHV Ab titers induced by the virus infection. Besides, a reduction of some alarmin release, i.e, uric acid and high-mobility group box1 protein (HMGB1), was observed. Accordingly, since alarmin liberation was related to the expression of autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), these autoAb also diminished. Moreover, PCR results indicated that TDO inhibition did not abolish viral replication. Furthermore, histological liver examination did not reveal strong pathologies, whereas mouse survival was hundred percent in control as well as in MHV-infected mice treated with LM10. Data presented in this work indicate that in spite of the various TDO actions already described, specific TDO blockage could also restrain some MHV actions, mainly suppressing autoimmune reactions. Such results should prompt further experiments with various viruses to confirm the possible use of a TDO inhibitor such as LM-10 to treat either viral infections or even autoimmune diseases triggered by a viral infection.