We are all familiar with SARS-CoV-2, the virus responsible for COVID-19 and the ongoing pandemic that has now seen nearly 800 million cases worldwide according to the World Health Organization. Most have also probably heard of SARS-CoV-2’s relatives, particularly SARS-CoV, which caused a smaller pandemic in 2003, and MERS-CoV, a rarer but more severe virus that continues to circulate mainly in western Asia and the Arabian Peninsula. However, these headline grabbers are also related to many other human coronaviruses (HCoVs) that cause more mild disease and are responsible for 15-30% of common cold cases worldwide (1). This range of disease severity caused by various HCoVs is an important area of study for virologists because it represents a sort of natural experiment; differences in pathogenesis between mild and severe HCoVs can yield insights about protective host immune responses and key virulence factors. However, determinants of disease severity in HCoVs remain poorly understood. Luckily, recent work by CAMB-MVP MD/PhD candidate Clayton Otter and colleagues in Susan Weiss’ lab has elucidated shared characteristics of common cold-associated HCoVs that may be predictive of infection outcomes and symptom severity in both mild and severe HCoV infections.
Subsets of HCoVs have been known to cause common colds for decades, but research of their infection dynamics was somewhat overlooked due to their mild nature. Interest in studying these non-lethal respiratory viruses has, of course, greatly increased since the onset of the SARS-CoV-2 pandemic. Clayton’s work in this study focuses on examining virulence factors, replication kinetics, and induced host immune responses in both mild and severe HCoVs to understand the marked differences in disease severity associated with these viruses.
To investigate the behavior of different HCoVs, the authors used a primary cell culture system with patient-derived nasal epithelial cells differentiated at an air-liquid interface.
Cells were cultured on transwell supports with specialized air-liquid interface (ALI) media on the basal face of the transwell and no media on the apical face. This unique approach allowed the authors to effectively model the environment of the nasal epithelium and upper airway, where primary infection and replication of HCoVs occurs.
Additionally, this system allowed for equilibration of nasal epithelial cultures at either 33°C or 37°C to further model the microenvironment of the upper and lower respiratory tract, respectively. Patient-derived ALI cultures were used to study replication dynamics and host immune response in the nasal epithelium following challenge with two common cold-associated HCoVs (HCoV-NL63 and HCoV-229E) and two severe HCoVs (SARS-CoV-2 and MERS-CoV).
Infection of ALI nasal epithelial cells showed that common-cold HCoVs are quickly cleared by the innate immune response after a rapid peak of initial replication. On the other hand, severe HCoVs replicate more slowly in initial time points, but eventually replicate strongly and reach a plateau; in other words, these viruses cannot be cleared by the immune response of nasal epithelial cells. Based on this difference in replication kinetics, Clayton hypothesized that common-cold HCoVs induce a robust host immune response in nasal epithelial cells, while severe HCoVs are capable of blunting or evading the host immune response. Indeed, bulk RNA-sequencing of infected nasal epithelial cultures showed that common-cold HCoVs induce strong interferon responses, a typical innate immune antiviral pathway. Interferons (IFNs) are cytokines that are produced after cytosolic pattern recognition receptors sense viral nucleic acids. Their production leads to the upregulation of a host of antiviral effectors called interferon stimulated genes (ISGs) capable of antagonizing viral life cycles at multiple stages. This suggests that the rapid clearance of common-cold HCoVs by nasal epithelial cells may depend on this antiviral pathway.
In contrast, SARS-CoV-2 only mildly induces the interferon response, whereas MERS-CoV infection does not trigger interferon production at all. This comparative lack of interferon response and ISG induction by these more severe HCoVs is likely what prevents nasal epithelial cells from resolving these infections. To test this more directly, Clayton performed another round of infection with HCoVs in ALI nasal epithelial cells, this time in conjunction with ruxolitinib treatment. Ruxolitinib is a small molecular inhibitor of JAK1/2 signaling that prevents transcription of ISGs downstream of interferon sensing at the cell surface, effectively blocking antiviral interferon responses. Ruxolitinib treatment led to increased replication of common-cold HCoVs in ALI cultures and prevented viral clearance, validating that restriction of these viruses by nasal epithelial cells depends on interferon signaling. Interestingly, ruxolitinib treatment had a minimal effect on viral kinetics of MERS-CoV and SARS-CoV-2 in ALI cultures. The authors expected this due to the inability of nasal epithelial cells to clear these severe HCoVs under normal infection conditions and the lack of strong interferon responses induced in these infections. Conversely, pre-treatment of ALI cultures with either IFNβ or IFNλ strikingly attenuated replication of both severe and common-cold HCoVs. This suggests that under normal conditions, severe HCoVs somehow antagonize the interferon response, facilitating increased replication.
Both SARS-CoV-2 and MERS-CoV encode accessory proteins that counteract the activity of cytosolic pattern recognition receptors that sense viral nucleic acids and mount IFN production. These virulence factors consist of the conserved non-structural protein nsp15 and an additional MERS-CoV accessory protein called NS4a. These proteins antagonize the interferon response by digesting or sequestering viral nucleic acids, respectively, thereby preventing receptors such as MDA5 from triggering IFN pathways. To test whether these virulence factors are necessary for SARS-CoV-2 and MERS-CoV replication in nasal epithelial cells, Clayton performed another round of infections with mutant versions of these viruses in which the relevant accessory proteins were deleted. Infection of ALI cultures with these mutant severe HCoVs led to robust induction of the interferon response and attenuated replication compared to wild type viruses. These data show that inactivation of the interferon response by severe HCoV accessory proteins is critical for their robust replication in the nasal epithelium.
Previous work by the Weiss group has shown that all assayed HCoVs except MERS-CoV preferentially replicate at 33°C - a temperature associated with the nasal cavity and upper airway - compared to 37°C, the typical temperature of the lungs or lower airway. In accordance with this, mild HCoVs tend to replicate only in the colder upper airway without ever penetrating into the lungs, leading to less severe disease in vivo. Based on the importance of interferon responses demonstrated in this manuscript, Clayton and colleagues hypothesized that nasal epithelial cells produce more robust IFN induction and ISG upregulation at 37°C. Indeed, interferon responses as measured by STAT phosphorylation and ISG protein levels are significantly upregulated when ALI cultures are infected at 37°C compared to 33°C. This stronger interferon response at warmer temperatures led to faster viral clearance of common-cold HCoVs, but again failed to clear SARS-CoV-2 infection, likely due to this virus’ ability to antagonize ISG induction. This also mirrors the in vivo situation, in which severe HCoVs maintain the ability to replicate in the warmer microenvironment of the lower airway and lung, leading to more extreme symptoms.
Of course, at this stage of the COVID-19 pandemic, there is no single SARS-CoV-2 virus, as innumerable variants have emerged due to the large number of total cases. Only a small subset of these variants are thoroughly characterized: typically those that become the most prevalent or “dominant” strain during a given surge in cases. The omicron variants are some of the most well studied novel strains of SARS-CoV-2 due to their extreme prevalence during the major wave of infections in the winter of 2021-2022, when the CDC abruptly and unscientifically reduced the recommended quarantine time for COVID patients (2). One of the defining characteristics of the various omicron strains was a penchant for replication in the upper respiratory tract, not unlike common-cold associated HCoVs. Given this clinical context, Clayton hypothesized that these SARS-CoV-2 variants would also replicate faster at 33°C rather than 37°C and induce a robust IFN response comparable to what was observed with common cold HCoVs. However, despite triggering a strong induction of ISGs and IFN secretion like common cold HCoVs, BA.1 replicated at comparable rates at each temperature and was not cleared by nasal epithelial cells at later time points. This suggests that BA.1 is less susceptible to IFN-mediated restriction than common cold HCoVs. Furthermore, even pre-treatment with IFNβ or IFNλ did not restrict replication of BA.1 or lead to viral clearance, suggesting that the BA.1 variant of SARS-CoV-2 is substantially less interferon-sensitive than ancestral strains overall. These data could potentially have important epidemiological implications, as novel SARS-CoV-2 variants may be growing more resistant to protective interferon responses that help to mitigate respiratory infections.
Clayton’s data show that interferon responses restrict the replication of common cold HCoVs. These viruses preferentially replicate in colder environments reminiscent of the nasal epithelium and upper airway. All of these phenotypes of mild HCoVs are similar to those observed in other common cold viruses such as human rhinovirus 16. This suggests that these shared characteristics are what drives the mild disease caused by these common cold viruses. On the other hand, severe HCoVs are not controlled by interferon responses in the nasal epithelium. Potentially lethal HCoVs encode accessory proteins that evade canonical interferon induction, and these virulence factors are indispensable for viral replication in epithelial cells throughout the airway. However, some severe HCoVs remain interferon sensitive, as pre-treatment with IFNβ or IFNλ showed. Additionally, recent clinical studies have shown that a stronger IFN response in the nasal epithelium is highly correlated with a more mild course of COVID-19 disease (3). Therefore, administration of these cytokines could potentially be therapeutically or prophylactically useful for treatment of the now omnipresent severe HCoVs. That said, Clayton’s analysis of the SARS-CoV-2 variant BA.1 suggests that more novel strains may be evolving their way out of this interferon sensitivity. These data highlight the importance of continuing to monitor and study SARS-CoV-2 variants of concern and the many insights that are available through research of more “mundane” common-cold associated HCoVs.
References
2:https://www.pbs.org/newshour/show/why-the-cdc-reduced-covid-quarantine-time-despite-omicrons-spread
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