Betacoronavirus internal protein: role in immune evasion and viral pathogenesis

Abstract

Betacoronaviruses express a small internal (I) protein that is encoded by the same subgenomic RNA (sgRNA) as the nucleocapsid (N) protein. Translation of the +1 reading frame of the N sgRNA through leaky ribosomal scanning leads to expression of the I protein. The I protein is an accessory protein reported to evade host innate immune responses during coronavirus infection. Previous studies have shown that the I proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus suppress type I interferon production by distinct mechanisms. In this review, we summarize the current knowledge on the I proteins of betacoronaviruses from different subgenera, with emphasis on its function and role in pathogenesis.

Keywords: coronavirus; immune evasion; internal protein; pathogenesis; virology.

Fig 1

Expression strategy of betacoronavirus internal protein. (A) Schematic diagram depicting the genome organization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is used as an example to illustrate the expression of the I protein (9b). (B) Schematic diagram showing the expression of the N and I proteins. Subgenomic RNA (sgRNA) expressing the N protein is depicted. Leaky ribosomal scanning happens when translation is not initiated at the start of codon of the N protein. Instead, translation is initiated at the start codon of 9b, leading to the expression of the protein. The relative abundance of the internal protein expression relative to the N protein is plausibly determined by the “strength” of the Kozak sequence for the initiation of N protein translation. The Bovine enteric coronavirus (BCoV) I protein:N protein expression has been previously determined as 1:2 at molar ratio (8). The levels of the Middle East respiratory syndrome coronavirus (MERS-CoV) I protein expression were detected in infected cells (1, 9). The relative abundance of N and I protein expression in infected cells is illustrated as a table. “+” indicates that expression of the protein was detected, “++” indicates peak expression, and “−“ indicates extremely low levels or not detectable expression.

Fig 2

Functions of SARS-COV-2 I protein and its interactions with the host. Coronaviruses interact with cellular receptors for entry. Upon uncoating, the viral genomic materials activate the host RNA-sensing pathway, resulting in the activation of MAVS. Downstream signaling of MAVS requires (i) the activation of nuclear factor κB (NF-κB) essential modulator (NEMO) for inducing NF-κB-dependent gene expression and (ii) the recruitment of TBK1 for IRF3-dependent transcriptional activation. The SARS-CoV-2 I protein (SARS2 I) has been reported to inhibit K63-linked ubiquitination of NEMO, leading to the suppression of NF-κB activation (3). SARS2 I also allosterically disrupts the interaction between HSP90 and TOM70, which is critical for the recruitment of TBK1 to the MAVS signaling complex for IRF3 activation (2, 45). The presence of SARS2 I leads to the downregulation of IRF3-dependent and NF-κB-dependent gene expressions including IFNs and cytokines. The interaction between SARS2 I with TOM70 may lead to mitochondrial dysfunction as TOM70 is known to mediate protein transport across the mitochondrial membrane (49, 50). In addition, host E3 ubiquitin ligases HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5 induce K48-linked ubiquitination of SARS2 I and lead to its degradation (47). The MERS-CoV I protein (MERS I) was reported to interact with HSP70. This prevents HSP70-dependent IKKε activation and hence suppression of IRF3 phosphorylation and IFN induction (1). This figure was created with BioRender.

Fig 3

Sequence alignments for I proteins of closely related betacoronaviruses. The I protein sequences of MHV-JHM and MHV-A59 (A), MERS-CoV and BatCoV-HKU4/5 (B) are aligned and compared. Amino acid sequences upstream of the start codon of the I proteins for MHV-JHM and MERS-CoV are illustrated as MHV-JHM (I*) in panel A and MERS-CoV (I*) in panel B, respectively. MHV-JHM (I*) and MERS-CoV (I*) are not expressed as part of the MHV-JHM and MERS-CoV I proteins, respectively, due to the presence of a stop codon in the sequence (highlighted in red). These result in the translation of MHV-JHM and MERS-CoV I proteins initiating at a position equivalent to the second start codon of MHV-A59 and BatCoV-HKU4/5 I proteins, respectively. These stop codons are not observed in MHV-A59 (A) or BatCoV-HKU4/5 (B) I proteins. MHV-JHM (I*) shares a high degree of similarity with the N-terminal part of the MHV-A59 I protein. MERS-CoV (I*) shares moderate similarity with the N-terminal parts of the BatCoV-HKU4/5 I proteins. The C-terminal part of the MERS-CoV I protein is also truncated compared to those of BatCoV-HKU4/5. “–” represents a gap in the sequences.