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 can also be read on my website† Here we discuss new data related to the ORF7a viral protein.
During the initial stages of cellular infection, SARS-CoV-2 releases a number of additional proteins to help suppress and evade our immune system. ORF7a is one such protein. Suppression of the innate immune system is necessary for the virus to establish an infection. The virus’s multi-day suppression of the immune system also contributes to the asymptomatic incubation period. During this time, the virus can be passed from person to person before anyone even knows they are infected. ORF7a is an important player in preventing the phosphorylation of a host protein called signal transducer and activator of transcription 2 (STAT2), which is essential for the induction of antiviral interferon-stimulated genes. New research highlights additional agents through which ORF7a contributes to the infectivity, pathogenesis and transmission of SARS-CoV-2. Published in nature communication† work by Timilsina et al. describes how ORF7a counteracts the protective effects of serine incorporator 5 (SERINC5), a host protein that blocks viral access to cells.
On the offensive: SERINC5
The antiviral functions of SERINC5 against retroviruses, including human immunodeficiency virus 1 (HIV-1) and mouse leukemia virus (MLV), are well documented. Still, little is known about its role in coronavirus infections. Timilsina and his colleagues wanted to fill this gap in our knowledge.
They first examined the expression levels of each member of the SERINC gene family — one to five — in lung tissue and Calu-3 lung cells. All except SERINC4 were abundantly expressed. Infection with SARS-CoV-2 did not affect the expression of SERINC1,2,3 and 5 in lung tissue or Calu-3 lung cells.
Next, the researchers examined whether any of the SERINC genes expressed in the lung tissue exhibit protective effects similar to those of SERINC5 in HIV-1. To do this, they produced pseudoviruses that replaced HIV-1’s external protein with the SARS-CoV-2 Spike protein — which the virus uses to bind to host receptors and enter cells. All SERINC proteins were absorbed into the SARS-CoV-2 Spike pseudovirions. When exposed to these SERINC-containing pseudovirions, lung and kidney cells were significantly less likely to become infected. SERINC5 was found to be particularly effective in reducing infectivity in both cell types (Figure 1). SERINC3 slightly reduced viral infectivity. SERINC1 and SERINC2 had no effect.
But retroviruses and coronaviruses are quite different from each other. First, they assemble in different parts of the host cell; retroviruses in the plasma membrane and coronaviruses in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The pseudoviruses the researchers generated may not accurately reflect what happens to coronaviruses. To remedy this, they performed the same experiments on both SARS-CoV-2 virus-like particles (VLPs) — non-infectious replicas of the virus — and infectious SARS-CoV-2. As before, SERINC5 successfully incorporated itself into the Spike protein and successfully reduced viral infectivity (Figure 2). SERINC3 was incorporated into the infectious SARS-CoV-2, but had a much smaller impact on viral infectivity.
The researchers also tested for the incorporation of SERINC5 into the Spike protein of several SARS-CoV-2 variants – Alpha (B.1.1.7), Beta (B.1.351), Gamma (P1) and Delta (B.1.617 ) – to ensure that any mutations in the Spike protein did not negate the protective effects on SERINC5. In all variants, SERINC5 continued to limit viral infectivity (Figure 3).
So SERINC5 gets incorporated into the Spike protein of SARS-CoV-2 and from there it manages to reduce the infection. But how exactly does it achieve this?
Timilsina et al. initially suspected that SERINC5 disrupts receptor binding, the first step in the process by which SARS-CoV-2 enters our cells. This happens when SARS-CoV-2 uses its Spike protein to bind to angiotensin-converting enzyme 2 receptors (ACE2) on the outside of our cells. Blocking this interaction would block the possibility of infection. To the researchers’ surprise, SERINC5 did not affect the interaction between the Spike protein and the ACE2 receptors of our cells.
Nor did SERINC5 interfere with the next stage of cellular infection – cleavage of the Spike protein into two parts.
Once the SARS-CoV-2 Spike protein is bound to ACE2 and cleaved, the remaining portion of the Spike protein inserts itself into the host cell’s membrane and pulls itself in, causing the two to fuse. Timilsina et al. found that, in the presence of SERINC5, Spike-mediated fusion with the host cell membrane was noticeably reduced. This suggests that SERINC5 blocks viral access by interfering with the fusion stage of infection. The exact mechanism by which it does this has yet to be determined.
Viral counterattack: ORF7a
HIV-1 has developed a way to parry the blow of SERINC5; it encodes a protein called Nef that prevents the antiviral protein from being incorporated into the nascent virions. What about SARS-CoV-2, is it launching its own counter-attacks?
The group of researchers turned to SARS-CoV-2 accessory proteins, which are known to block host antiviral genes and suppress the immune response. They addressed ORF7a. During infection with SARS-CoV-2, ORF7a moves to the endoplasmic reticulum and Golgi apparatus of the host cell. This is the area where new viral particles are assembled and, by extension, where SERINC5 can incorporate itself into the nascent Spike protein.
Timilsina et al. tested their hypothesis by exposing lung and kidney cells to a knockout strain of SARS-CoV-2 that does not contain ORF7a. They compared the results with infection with unmutated wild-type SARS-CoV-2. The amount of SERINC5 packaged in the nascent virions was significantly increased in the ORF7a knockout strain. Higher levels of SERINC5 in the knockout strain were reflected by overall poorer viral infectivity. Reintroduction of ORF7a into the knockout strain resulted in infectivity. These findings confirm that, in the context of SARS-CoV-2 infection, ORF7a acts to prevent the incorporation of SERINC5 into emerging viral particles.
Timilsina and his colleagues propose two methods by which ORF7a inhibits SERINC5. One method is to avoid packaging SERINC5 into the nascent virion particles in the first place. The second method is done within the viral particles. The researchers suggest that ORF7a forms a complex with the Spike protein and with SERINC5 that ultimately blocks SERINC5 from restricting viral access. They were able to confirm that the SARS-CoV-2 Spike protein, ORF7a and SERINC5 all converge and interact in the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) (Figure 4). The nuances of how this complex undermines SERINC5 remain unknown. Analogous to HIV-1, the authors hypothesize that SERINC5 can alter the structure of the Spike protein and that ORF7a binds to the Spike protein to prevent such changes. Future research should aim to resolve this unknown through in-depth structural analysis.
Mutations of accessory proteins are not rare and a number of naturally occurring deletions have been detected in ORF7a; does any of them undermine his ability to counter-attack against SERINC5?
Timilsina et al. tested four naturally occurring deletions of ORF7a — Δ9nt, 18nt, Δ57nt and Δ96nt — isolated from clinical samples from infected patients. The ability to block SERINC5 was preserved in all four SARS-CoV-2 variants with naturally occurring ORF7a mutations. There was little to no difference in the degree of SERINC5 restriction between the ORF7a mutations and the wild-type ORF7a.
Of more general concern, inhibition of ORF7a will attenuate SARS-CoV-2 replication, allowing our natural cellular defenses to be more effective at fighting off the virus. This work by Timilsina et al. adds another reason to consider ORF7a as an important antiviral target for future drug development.
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