Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Abstract

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8

“…We thus aimed to determine how the lack of specific accessory proteins impacts SARS-CoV-2 transmission in our model, and focused on two accessory proteins: ORF8, because it has mutations defining the Alpha, Gamma, and Delta variants, and, ORF6, which lacks mutations in the variants utilized in this study but is mutated in some Omicron variants (i.e. BA.2, BA.4), in addition to being one of the best-characterized SARS-CoV-2 accessory proteins 4, 42–44 . Both of these recombinant viruses have been characterized in cultured cells and in adult K18-hACE2 mice 45 , revealing reduced viral titers in vitro , but similar lung titers in vivo .…”

On the Evolutionary Trajectory of SARS-CoV-2: Host Immunity as a Driver of Adaptation in RNA Viruses

“…The Alpha variant is associated with even lower activation of IRF-3 compared to the ancestral strain, as well as reduced activation of nuclear factor kappa B (NF-κB), which is also involved in IFN-β transcription [ 49 , 50 ]. Upregulation of ORF6 in the Alpha variant likely also contributes to its enhanced transmission and infectivity [ 51 , 52 ]. Interestingly, the recently emerged Omicron subvariants BA.2 and BA.4 harbour a D61L mutation in ORF6, which attenuates its capacity to interact with IRF-3 nuclear import factors, reducing IFN evasion [ 52 ].…”

“…Upregulation of ORF6 in the Alpha variant likely also contributes to its enhanced transmission and infectivity [ 51 , 52 ]. Interestingly, the recently emerged Omicron subvariants BA.2 and BA.4 harbour a D61L mutation in ORF6, which attenuates its capacity to interact with IRF-3 nuclear import factors, reducing IFN evasion [ 52 ]. This mutation is no longer present in the currently circulating BA.5 subvariant.…”

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing

“…We next explored the ability of scCoVseq to quantify sgmRNAs as compared to previously published methods. To do this, we aggregated (to “pseudobulk” profiles) scRNA-Seq data of ACE2-expressing A549 cells infected with recombinant SARS-CoV-2 (rSARS-CoV-2) ORF6 M58R, an attenuated mutant that allows for high MOI infection without excessive cell death (45). In parallel, triplicate bulk RNA-Seq libraries were prepared from these cultures and quantified with periscope, a previously published method to quantify SARS-CoV-2 sgmRNAs in bulk RNAseq data using partial alignments of viral reads to TRS and ORF sequence(42).…”

“…For experiments involving A549-ACE2 cells, a recombinant SARS-CoV-2 (rSARS-CoV-2) virus, rSARS-CoV-2 ORF6 M58R, was used as described elsewhere (45). All viral stocks were grown in Vero E6 cells as previously described and validated by genome sequencing (35).…”