Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19.

Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the recently identified Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV).

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Coronaviruses: An Overview of Their Replication and Pathogenesis

Epstein-Barr virus.

Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syndrome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expression. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple subgenomic RNA species, and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed by the enormous size of the coronavirus genome. This review summarizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly.

The molecular biology of coronaviruses.

The SARS-CoV-2 pandemic and its unprecedented global societal and economic disruptive impact has marked the third zoonotic introduction of a highly pathogenic coronavirus into the human population. Although the previous coronavirus SARS-CoV and MERS-CoV epidemics raised awareness of the need for clinically available therapeutic or preventive interventions, to date, no treatments with proven efficacy are available. The development of effective intervention strategies relies on the knowledge of molecular and cellular mechanisms of coronavirus infections, which highlights the significance of studying virus-host interactions at the molecular level to identify targets for antiviral intervention and to elucidate critical viral and host determinants that are decisive for the development of severe disease. In this Review, we summarize the first discoveries that shape our current understanding of SARS-CoV-2 infection throughout the intracellular viral life cycle and relate that to our knowledge of coronavirus biology. The elucidation of similarities and differences between SARS-CoV-2 and other coronaviruses will support future preparedness and strategies to combat coronavirus infections.

/pdf/coronavirus-biology-and-replication-implications-for-sars-47nwmem9di.pdf

Coronavirus biology and replication: implications for SARS-CoV-2.

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) causes a recently emerged human disease associated with pneumonia. The 5′ end two-thirds of the single-stranded positive-sense viral genomic RNA, gene 1, encodes 16 mature proteins. Expression of nsp1, the most N-terminal gene 1 protein, prevented Sendai virus-induced endogenous IFN-β mRNA accumulation without inhibiting dimerization of IFN regulatory factor 3, a protein that is essential for activation of the IFN-β promoter. Furthermore, nsp1 expression promoted degradation of expressed RNA transcripts and host endogenous mRNAs, leading to a strong host protein synthesis inhibition. SCoV replication also promoted degradation of expressed RNA transcripts and host mRNAs, suggesting that nsp1 exerted its mRNA destabilization function in infected cells. In contrast to nsp1-induced mRNA destablization, no degradation of the 28S and 18S rRNAs occurred in either nsp1-expressing cells or SCoV-infected cells. These data suggested that, in infected cells, nsp1 promotes host mRNA degradation and thereby suppresses host gene expression, including proteins involved in host innate immune functions. SCoV nsp1-mediated promotion of host mRNA degradation may play an important role in SCoV pathogenesis.

https://www.pnas.org/content/pnas/103/34/12885.full.pdf

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation.

Coronaviruses are enveloped RNA viruses that cause gastrointestinal and upper respiratory tract illnesses in animals and humans (65, 83). These range in severity from very serious neonatal enteritis in domestic animals to the common cold and severe acute respiratory syndrome in humans (24, 35). Mouse hepatitis virus (MHV), a prototypic coronavirus, causes various diseases, including hepatitis, enteritis, and encephalitis in rodents (19, 83). MHV contains a 32 kb-long, positive-sense, single-stranded RNA genome (37, 38, 63) that carries 11 open reading frames (ORFs). Those ORFs are expressed through the production of a genomic-size mRNA and six to eight species of subgenomic mRNAs (36, 39). MHV mRNA 1 encodes the 5′-most gene, the 22-kb-long gene 1, which contains two large overlapping ORFs, ORFs 1a and 1b (8, 38). A ribosomal frameshift that occurs at the 3′ end of ORF1a results in the ORF 1a and 1b genes being translated as a large polyprotein (11), which afterward is processed into smaller, mature nonstructural proteins (a total of ∼16 proteins) by virus-encoded proteinases (9, 10, 23, 46, 47).

Some of the known functions of the mature gene 1 proteins include RNA polymerase, helicase, and proteinases, but the purposes of most of the other gene 1-encoded proteins are unidentified (28, 38). Analyses of MHV-infected cells demonstrated that the N-terminal cleavage product of gene 1, p28, is detected in the cytoplasm (7) and in cytoplasmic membrane fractions containing viral RNA, helicase and nucleocapsid (N) protein, suggesting that p28 is a component of the putative MHV replication complex (77). However, the exact role of p28 during MHV infection remains unclear.

Many viruses manipulate the host's cell cycle regulation in order to further their own replication (reviewed in reference 61). Small DNA tumor viruses, such as simian virus 40 (21), adenovirus (34), and human papillomavirus (54), encode proteins that promote cells to enter the S phase. In contrast, herpesviruses, a group of large DNA viruses which encode their own DNA polymerases, generally block cell cycle progression in the G0/G1 phase during lytic infection cycles (reviewed in reference 26). Compared to the number of reports of cell cycle arrest caused by DNA viruses, few reports of RNA virus-induced cell cycle arrest exist. Reovirus infection has been known, for a long time, to inhibit cellular DNA synthesis (20, 29), but not until recently was it shown that the viral σ1s nonstructural protein mediates reovirus-induced inhibition of cell proliferation by arresting host cells in the G2/M phase of the cell cycle (68, 69). Human immunodeficiency virus type 1 infection also induces cell cycle arrest in the G2/M phase (30); sole expression of the accessory gene product Vpr is sufficient for inducing G2/M cell cycle arrest (70, 72). Vpr-mediated cell cycle arrest apparently favors human immunodeficiency virus type 1 replication, because the long terminal repeat is most active and the expression of viral genome is optimal in the G2 phase of cell cycle (27). Cell cycle perturbations also are seen in cells infected with the paramyxovirus simian virus (41), measles virus (51, 56), and MHV (16).

Cyclins and cyclin-dependent kinases (Cdks) form complexes and play important regulatory roles in controlling cell cycle progression through the G1, S, G2, and M phases (reviewed in references 58 and 67). G1 phase progression requires the activity of cyclin D-Cdk4/6 complexes, and cyclin E-Cdk2 activity is necessary for the G1-S transition. These G1 cyclin-Cdk complexes regulate the cell cycle through phosphorylation of the retinoblastoma protein (pRb). In the quiescent G0 phase, pRb is unphosphorylated; it then is sequentially hypophosphorylated by cyclin D-Cdk4/6 complexes in early G1 phase and hyperphosphorylated by cyclin E-Cdk2 complex in mid- to late G1 (48). It remains hyperphosphorylated in the S, G2, and M phases of cycling cells (22). In its active, hypophosphorylated state, pRb is a transcriptional repressor when bound to the E2F family of transcription factors. Functionally pRb is inactivated when it is hyperphosphoryled in late G1, which results in the release of E2F and allows the transcription of genes that are important for DNA synthesis (reviewed in reference 84). Activities of G1 cyclin-Cdk complexes are regulated by Cdk inhibitors (CKIs), which can be grouped into two families (reviewed in reference 76). The INK4 family proteins bind to Cdk4 and Cdk6, thus blocking cyclin D-Cdk4/6 activities (reviewed in reference 75). CKIs of Cip/Kip family contain p21Cip1, p27Kip1, and p57Kip2, which are potent inhibitors of cyclin E- and A-dependent Cdk2 (reviewed in reference 55). The tumor suppressor p53 exerts its antiproliferative effect by activating p21Cip1 (25); however, p21Cip1 also can be activated by p53-independent mechanisms (78).

The present study demonstrated that expression of MHV p28 had a growth-inhibitory function on the cultured cells in which it was expressed. Expression of p28 induced G0/G1 cell cycle arrest, which was mostly likely due to an increase in the hypophosphorylated form of pRb. The cell cycle block occurred together with the accumulation of p53 and p21Cip1, suggesting that p53-mediated activation of p21Cip1 was one mechanism for p28-induced growth arrest. This is the first demonstration that the expression of an RNA viral nonstructural protein can specifically arrest the cell cycle in the G0/G1 phase.

Hideyuki Kubo

Papers

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation.

Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase