SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98

Abstract

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6’s interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6’s disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.

Coronavirus, the King Who Wanted More Than a Crown: From Common to the Highly Pathogenic SARS-CoV-2, Is the Key in the Accessory Genes?

“…A deletion of 27 a.a, which maps to a putative signal peptide within the ORF7a protein, has recently been reported in two SARS-CoV-2 isolates from two separate patients (Holland et al, 2020). This large deletion of the ORF7a is proposed to prevent redundant functions on inhibition of cellular protein synthesis also found associated with the ORF6 protein that, as we have seen above, interacts with the mRNA export proteins NUP98 and RAE1 and may inhibit cellular translation (Addetia et al, 2020(Addetia et al, , 2021. The a.a sequence of the SARS-COV-2 ORF7a protein is very close to the ORF7a of bat-SL-CoV-RaTG13 ORF7a like that of the Pangolin-CoV-2019, but much more distant than that of the bat-SL- 97.5,and 40% a.a identity,respectively).…”

“…However, an upregulation of several genes, all involved in the NF-kB pathway, was noted after infection with these variants in comparison to the reference strain, suggesting that the truncated ORF6 proteins could play a role in the inflammatory host-response (Xia et al, 2020). Lately, the SARS-CoV-2 ORF6 protein function as a potent IFN antagonist (Yuen et al, 2020;Kimura et al, 2021) and also was found to block nuclear import of a wide range of host factors through interactions with RAE1 and NUP98 (Addetia et al, 2021).…”

How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System

“…The associations suggested by these PPI analyses need to be validated by further experiments. To date, only Orf6 has been shown to interrupt the host nucleocytoplasmic trafficking [75][76][77][78] and to induce an aberrant distribution of Nups (Nup98 and Rae1) [76]. Xia et al found that Orf6 binds to importin-α1 to block the nuclear translocation of IRF3, resulting in an impaired type I IFN production in the HEK293T cell line [78].…”

“…Nevertheless, these α-importins may not be the direct key players because their overexpression failed to rescue an Orf6-dependent blockade of green fluorescent protein (GFP)-tagged STAT1 nuclear import. Interestingly, Addetia et al reported that the SARS-CoV-2 Orf6 interactions with Nup98 and Rae1 were much stronger than those of SARS-CoV-1 Orf6, suggesting the strong IFN antagonism triggered by SARS-CoV-2 contributes to the high prevalence of asymptomatic cases of SARS-CoV-2 infection [77].…”

“…These findings, that is, the aberrant localization of Nup98 and Rae1 and the nuclear saturation of hnRNPA1, suggest that the Orf6-Nup98-Rae1 interaction can block mRNA export. Addetia et al later found that SARS-CoV-2-infected cells exhibited a nuclear mRNA accumulation that could be mediated by Orf6 [77]. Recently, Zhang et al reported that SARS-CoV-2 non-structural protein 1 (Nsp1) halted the host gene expression by blocking an Nxf1-Nxt1-mediated mRNA export [83].…”