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 the major inhibitor of interferon production in both SARS-CoV and SARS-CoV-2. SARS-CoV-2 ORF6 was recently shown to co-purify with the host mRNA export factors Rae1 and Nup98. Here, we demonstrate SARS-CoV-2 ORF6 strongly represses protein expression of co-transfected reporter constructs and imprisons host mRNA in the nucleus, which is associated with its ability to co-purify with 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. We further identify an ORF6 mutant containing a 9-amino acid deletion, ORF6 {Delta}22-30, in multiple SARS-CoV-2 clinical isolates that can still downregulate the expression of a co-transfected reporter and interact with Rae1 and Nup98. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly co-purifies 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 pre-symptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. ImportanceSARS-CoV-2, the causative agent of COVID-19, is an RNA virus with a large genome that encodes accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. One of SARS-CoV-2s accessory proteins, ORF6, was recently shown to co-purify with two host proteins, Rae1 and Nup98, involved in mRNA nuclear export. We demonstrate SARS-CoV-2 ORF6 interaction with these proteins is associated with reduced expression of a reporter protein and accumulation of poly-A mRNA within the nucleus. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and co-purifies with Rae1 and Nup98 compared to SARS-CoV ORF6. The ability of SARS-CoV-2 ORF6 to more strongly disrupt nucleocytoplasmic transport than SARS-CoV ORF6 may partially explain critical differences in clinical presentation between the two viruses.
ABSTRACT
Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3, and Th1 chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-{kappa}B signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.
ABSTRACT
Despite the continued spread of SARS-CoV-2 and emergence of variants of concern (VOC) that are capable of escaping preexisting immunity, therapeutic options are underutilized. In addition to preventing severe disease in high-risk patients, antivirals may contribute to interrupting transmission chains. The FDA has granted emergency use authorizations for two oral drugs, molnupiravir and paxlovid. Initial clinical trials suggested an efficacy advantage of paxlovid, giving it a standard-of-care-like status in the United States. However, recent retrospective clinical studies suggested a more comparable efficacy of both drugs in preventing complicated disease and case-fatalities in older adults. For a direct efficacy comparison under controlled conditions, we assessed potency of both drugs against SARS-CoV-2 in two relevant animal models; the Roborovski dwarf hamster model for severe COVID-19 in high-risk patients and the ferret model of upper respiratory tract disease and transmission. After infection of dwarf hamsters with VOC omicron, paxlovid and molnupiravir were efficacious in mitigating severe disease and preventing death. However, a pharmacokinetics-confirmed human equivalent dose of paxlovid did not significantly reduce shed SARS-CoV-2 titers in ferrets and failed to block virus transmission to untreated direct-contact ferrets, whereas transmission was fully suppressed in a group of animals treated with a human-equivalent dose of molnupiravir. Prophylactic administration of molnupiravir to uninfected ferrets in direct contact with infected animals blocked productive SARS-CoV-2 transmission, whereas all contacts treated with prophylactic paxlovid became infected. These data confirm retrospective reports of similar therapeutic benefit of both drugs for older adults, and reveal that treatment with molnupiravir, but not paxlovid, may be suitable to reduce the risk of SARS-CoV-2 transmission.