This article is an extension of our series on: immunosuppression by SARS-CoV-2† The series has since been published as a book, Natural immunity and Covid-19: what it is and how it can save your life† It is also available to continue reading my website† Here we discuss new data related to the ORF7a viral protein. Click for Part 1†
SARS-CoV-2’s success as a virus largely depends on its ability to suppress and evade our immune system. Usually, our immune system encounters an invading microbe and immediately springs into action. But SARS-CoV-2 is a master at flying under the radar and staying hidden from our innate immune response. It also evolves rapidly to evade our adaptive immune response.
Interferons – a group of signaling proteins produced by cells in response to microbial threat – are key to both the innate and adaptive immune responses. In a previous article, we set out how ORF7a inhibits the induction of interferon-stimulated genes by preventing the phosphorylation of STAT2. This hinders our interferon response, making it more difficult for our immune system to mount a successful counteroffensive. More recently we have described the discovery that ORF7a also blocks the antiviral function of a host protein called SERINC5, which would otherwise help prevent viral entry into cells.
A study by a group of researchers based at The Fifth Affiliated Hospital at Sun Yat-sen University emphasizes yet another immune evasive capability of ORF7a: the suppression of CD14+ monocytes. In addition, Zhou et al. suggest that ORF7a contributes to one of the most troubling aspects of Covid-19, an over-aggressive release of pro-inflammatory molecules. These molecules are called cytokines and their overexpression can lead to the life-threatening systemic inflammatory syndrome known as ‘cytokine storm’.
Covid-19 Severity: Monocytes and Inflammation
Most people who contract Covid-19 will have only mild or no symptoms. But there is a subgroup of patients who develop serious disease – with over half a billion confirmed cases, this subgroup is quickly getting very large. Severe cases are generally characterized by an overactive inflammatory response associated with elevated cytokine levels, a decrease in white blood cell counts, and infiltration of macrophages and monocytes into various tissues. Monocytes, in particular, appear to play an important role in the hyperinflammation seen in severe cases of Covid-19.
Monocytes are large white blood cells that circulate throughout the body through the bloodstream, watching out for any microbial threats. They recognize microbes through pattern recognition receptors (PRRs) that line their surface. These receptors pick up age-old molecular patterns typical of pathogens — be they viral, bacterial, fungal or parasitic. Once they spot a pathogen, they migrate to the affected area and help stimulate the inflammatory response by producing cytokines. Monocytes can also differentiate into two other types of immune cells: phagocytes, which engulf and destroy microbes, and dendritic cells, which supply T cells with antigens to stimulate a more specific immune response. As such, suppression of monocytes can lead to all kinds of domino-immune dysregulation.
While aberrant inflammation and monocyte upregulation had been associated with worse Covid-19 outcomes, the viral proteins responsible for this excess inflammation remained largely unknown.
ORF7a: New structural insights
Certain proteins contain immunoglobulin-like (Ig-like) molecular structures. These play a crucial role in modulating interactions in the immune system. Usually these are host proteins, such as antibodies. Certain viruses have evolved proteins with similar structures to help them hijack the host’s immune system.
Zhou et al. scanned SARS-CoV-2 proteins for Ig-like structures and noted that ORF7a contains an Ig-like ectodomain – the portion of a protein that extends from the surface and initiates contact with other proteins and cells. The ORF7a ectodomain consists of seven beta strands (β strands) forming two connected beta sheets (β sheets) (Figure 1). The resulting structure looks like a hand, with the palm facing inward. It is the “fingers” of this hand that eventually reach out and bind to other proteins.
ORF7a contains an Ig-like ectodomain, but does it actually interact with host immune cells? To find out, the researchers exposed human peripheral blood mononuclear cells (PBMCs) — lymphocytes and monocytes — from healthy donors to SARS-CoV-2 ORF7a. They found that ORF7a binds to CD14+ monocytes with a high degree of efficiency. It also binds to lymphocytes, but much weaker.
SARS-CoV-1, the virus responsible for the SARS outbreak in 2003, also has an ORF7a protein. The sequence similarity between the two is 87% and they also share a very similar structure. Yet the ORF7a protein of SARS-CoV-1 binds only weakly to monocytes. The researchers used this difference in binding affinity to identify the precise structural features of SARS-CoV-2 ORF7a that allow it to bind so efficiently. They found that the two ORF7a proteins differ from each other in the distribution of the binding site residues. Compared to SARS-CoV-1 ORF7a, the binding residues of SARS-CoV-2 ORF7a were all located on the larger beta sheet (strands A, G, F and C). None were on the smaller, three-stranded beta sheet (strands D, E, and B). This implies that the residues of the key bonds are located on the larger beta sheet (Figure 2).
ORF7a suppresses monocytes
After determining that ORF7a can bind to monocytes and lymphocytes, the researchers examined the effects of this interaction. They co-incubated SARS-CoV-2 ORF7a with human monocytes and lymphocytes for a period of 24 hours. Zhou et al. then measured the expression levels of human leukocyte antigen (HLA) surface molecules on the immune cells.
The HLA system is used to present antigens to other cells and is a critical part of the adaptive immune response. It can be divided into two classes. Firstly, those proteins that help to move antigen fragments from the inside of a cell to the cell surface. This signals the immune system that a cell is infected and needs to be destroyed, slowing the spread of the pathogen. This class consists of HLA-A, HLA-B and HLA-C. The second class consists of HLAs that present antigens found outside the cell to helper T cells. The T helper cells then stimulate the production of B cells, which secrete antibodies specific to the microbial threat. This class includes HLA-DR, HLA-DP, and HLA-DQ.
The researchers noticed a significant decrease in the expression of the second kind of HLAs on the surface of CD14+ monocytes – about 30% lower than normal. There was no difference in the expression of HLA-A/B/C. The expression of HLAs on the surface of lymphocytes remained unchanged.
Thus, ORF7a reduces the amount of HLA receptors on the surface of CD14+ monocytes, hampering their ability to signal for additional support from other immune cells. Zhou et al. conclude that this is likely an immune escape tactic on behalf of SARS-CoV-2 – essentially, giving it more time to spread undetected. The precise mechanism by which the ORF7a ectodomain modulates the antigen-presenting ability of CD14+ monocytes remains to be determined.
ORF7a causes inflammation
As mentioned, hyperinflammation is a hallmark of Covid-19. In a previous article we discussed how SARS-CoV-2 can infect monocytes, leading to a form of cell death called pyroptosis and the sudden release of a mass of pro-inflammatory cytokines. Findings by Zhou et al. implicate ORF7a as an additional source of monocyte-mediated inflammation.
To study the effects of ORF7a on inflammation, the team of scientists tested blood samples for markers of inflammation. They found that co-incubation with ORF7a caused a strong increase in the production of pro-inflammatory cytokines. More specifically, they noted that co-incubation led to the upregulation of those cytokines most associated with cytokine storms – IL-6, IL-1β, IL-8 and TNF-α. The spike in cytokine production implies that ORF7a monocyte immunomodulation may be a contributing factor to the hyperinflammation seen in severe Covid-19 cases.
Interestingly, the cytokine profiles differed depending on the donor. Some blood samples had a more intense cytokine response than others. This indicates that there may be genetic differences that determine ORF7a interaction and, by extension, disease outcome. Future research should focus on locating the factors responsible for the variation in cytokine response.
ORF8, another SARS-CoV-2 accessory protein, is also known to cause inflammation. In this case, the process is highly focused, with: Mimic ORF8 one of the most potent triggers for our immune system’s inflammatory responses, interleukin-17.
The question arises, is the inflammation caused by SARS-CoV-2 infection just an unfortunate side effect, or could it be in favor of viral replication? One observation supporting the hypothesis that inflammation may contribute to the success of the virus is that the receptor for viral entry, angiotensin converting enzyme 2 (ACE2), is induced as part of the inflammatory response in endothelial cells and other cells. A release of inflammatory substances can therefore be advantageous, and not entirely accidental.
The ability of ORF8 and ORF7a to induce an inflammatory response on their own favors the favorable hypothesis.
Message to take home
This work by Zhou et al. opens the door to a new drug development target, ORF7a. It also adds to the growing list of reasons to shift our focus to the entire SARS-CoV-2 genome, not just the Spike protein. There are many additional proteins that contribute to the virus’ ability to evade and suppress our immune system; the sooner we learn about their role in infection, the sooner we can start making effective anti-covid drugs.
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