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This article is an extension of our series on SARS-CoV-2 immune suppression. Since then, the series has 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 read on my website. Here, we talk about new data on the ORF7a viral protein.
During the early stages of cell infection, SARS-CoV-2 releases a number of accessory proteins to help suppress and evade our immune system. ORF7a is one of these proteins. Suppression of the innate immune system is necessary for the virus to establish infection. Multi-day suppression of the immune system by the virus also contributes to the asymptomatic incubation period. During this period, the virus can be transmitted from person to person before anyone realizes that they have been infected. ORF7a is a key player in preventing the phosphorylation of a host protein called signal transducer and transcription activator 2 (STAT2), which is critical for the induction of antiviral-stimulated genes. New research highlights additional means by which ORF7a contributes to the infectivity, pathogenesis, and transmission of SARS-CoV-2. Published in Nature Communications, the work of Timilsina et al. details how ORF7a counteracts the protective effects of serine 5 incorporator (SERINC5), a host protein that blocks viral entry into cells.
On the offensive: SERINC5
The antiviral functions of SERINC5 against retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), are well documented. However, little is known about its role in coronavirus infections. Timilsina and her colleagues set out 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 expressed themselves abundantly. SARS-CoV-2 infection did not affect the expression of SERINC1,2,3 and 5 in lung tissue or Calu-3 lung cells.
The researchers then looked at whether any of the SERINC genes expressed in lung tissue had protective effects similar to those seen with SERINC5 in HIV-1. To do so, they produced pseudoviruses that replaced the outer protein of HIV-1 with the Spike SARS-CoV-2 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 pseudovirons, lung cells and kidney cells were significantly less likely to become infected. SERINC5 has been shown to be especially effective in reducing infectivity in both cell types (Figure 1). SERINC3 modestly reduced viral infectivity. SERINC1 and SERINC2 had no effect.
FIGURE 1. Relative infectivity of lung cells (Calu-3) and kidney cells (293T-hACE2) when exposed to … [+] Spike pseudovirus SARS-CoV-2 with (SERINC1, 2, 3 and 5) and without (EV) SERINC packaged in Spike pseudovirions. SERINC5 potently blocks Spike SARS-CoV-2 protein-mediated entry into lung and kidney cells.
BY: TIMILSINA ET AL. 2022
But retroviruses and coronaviruses are quite different from each other. On the one hand, they are grouped in different parts of the host cell; retrovirus in the plasma membrane and coronavirus in the intermediate compartment of the endoplasmic reticulum-Golgi (ERGIC). The pseudoviruses generated by the researchers may not accurately represent what happens with coronaviruses. To solve this, they performed the same experiments with both SARS-CoV-2 virus-like particles (VLPs) —not-infectious replicas of the virus — and SARS-CoV-2 infectious particles. As before, SERINC5 was successfully incorporated into the Spike protein and successfully reduced viral infectivity (Figure 2). SERINC3 was incorporated into the SARS-CoV-2 infectious, but had a much smaller impact on viral infectivity.
FIGURE 2. Relative infectivity of lung cells (Calu-3) and kidney cells (293T-hACE2) when exposed to … [+] SARS-CoV-2 VLP and infectious viruses with (SERINC3 and 5) and without (EV) SERINC packaged in Spike protein. Again, SERINC5 potently blocks Spike SARS-CoV-2-mediated entry into lung and kidney cells.
BY: TIMILSINA ET AL. 2022
The researchers also tested the incorporation of SERINC5 into the Spike protein of different SARS-CoV-2 variants —Alpha (B.1.1.7), Beta (B.1.351), Gamma (P1), and Delta (B.1.617). – to Make sure no mutations in the Spike protein denied the protective effects of SERINC5. In all variants, SERINC5 continued to restrict viral infectivity (Figure 3).
FIGURE 3. Relative infectivity of kidney cells (293T-hACE2) when exposed to different variants of … [+] Spike SARS-CoV-2 pseudovirons with and without (EV) SERINC packaged in Spike protein.
BY: TIMILSINA ET AL. 2022
Thus, SERINC5 is incorporated into the Spike protein of SARS-CoV-2 and, from there, manages to reduce the infection. But how do you do that?
Timilsina et al. initially suspected that SERINC5 interferes with receptor binding, the first step in the process by which SARS-CoV-2 enters our cells. This is when SARS-CoV-2 uses its Spike protein to bind to angiotensin 2 converting enzyme (ACE2) receptors on the outside of our cells. Blocking this interaction would block the possibility of infection. To the researchers’ surprise, SERINC5 had no impact on the interaction between the Spike protein and the ACE2 receptors in our cells.
SERINC5 also did not interfere with the next stage of cell infection: the division of the Spike protein into two parts.
Once the Spike SARS-CoV-2 protein has bound to the ACE2 and cleaved, the remaining section of the Spike protein is inserted into the membrane of the host cell and enters, fusing. both. Timilsina et al. found that in the presence of SERINC5, Spike-mediated fusion to the host cell membrane was markedly reduced. This suggests that SERINC5 blocks viral entry by interfering with the stage of infection spread. It remains to be determined the precise mechanism by which it does so.
Viral counterattack: ORF7a
HIV-1 has developed a way to stop the blow given by SERINC5; encodes a protein called Nef that prevents antiviral protein from being incorporated into germ virions. What about SARS-CoV-2, does it have any counterattacks of its own?
The group of researchers used the SARS-CoV-2 accessory proteins, which are known to block host antiviral genes and suppress the immune response. They were perfected in ORF7a. During SARS-CoV-2 infection, ORF7a moves into the endoplasmic reticulum and the Golgi apparatus of the host cell. This is the area where new viral particles are grouped and, by extension, where SERINC5 can be incorporated into the nascent Spike protein.
Timilsina et al. they tested their hypothesis by exposing lung and kidney cells to a SARS-CoV-2 elimination strain that does not contain ORF7a. They compared the results with non-mutated wild-type SARS-CoV-2 infection. The amount of SERINC5 packaged in germ virions was significantly increased in the ORF7a elimination strain. Higher levels of SERINC5 in the elimination strain were reflected by lower overall viral infectivity. The reintroduction of ORF7a into the knock-out strain saved the infectivity. These findings confirm that, in the context of SARS-CoV-2 infection, ORF7a works to prevent the incorporation of SERINC5 into nascent viral particles.
Timilsine and colleagues propose two methods by which ORF7a inhibits SERINC5. One method is to prevent SERINC5 from packing into nascent virion particles in the first place. The second method goes inside the viral particles. The researchers suggest that ORF7a forms a complex with the Spike protein and with SERINC5 that eventually blocks SERINC5 from restricting viral entry. They were able to confirm that the Spike SARS-CoV-2, ORF7a, and SERINC5 proteins bind to and interact in the intermediate compartment of the endoplasmic reticulum-Golgi (ERGIC) (Figure 4). The nuances of how this complex undermines SERINC5 remain unknown. By analogy with HIV-1, the authors hypothesize that SERINC5 may alter the structure of the Spike protein and that ORF7a binds to the Spike protein to prevent these changes. Future research should aim to resolve this unknown through in-depth structural analysis.
FIGURE 4. Location of the Spike SERINC5, ORF7a and SARS-CoV-2 protein in ERGIC.
BY: TIMILSINA ET AL. 2022
Accessory protein mutations are not uncommon and a number of natural suppressions have been detected in ORF7a; did any of them undermine their ability to mount a counterattack against SERINC5?
Timilsina et al. tested four natural ORF7a suppressions (Δ9nt, Δ18nt, Δ57nt, and Δ96nt) isolated from clinical samples from infected patients. The ability to block SERINC5 was maintained in all four SARS-CoV-2 variants with natural ORF7a mutations. There was little or no difference in the extent of the SERINC5 restriction between ORF7a and wild-type ORF7a mutations.
Implications
Of more general importance is that inhibition of ORF7a will weaken SARS-CoV-2 replication, allowing our natural cellular defenses to be more effective in preventing the virus. This work by Timilsina et al. adds another reason to consider ORF7a an important antiviral target for future drug development.