The coronavirus known as SARS-CoV-2, the causative agent of the COVID-19 pandemic, induces a puny innate immune response compared other respiratory viruses such as the flu. In a preprint posted on bioRxiv on May 12, researchers have shown in cell culture that a viral protein called open reading frame 3b (ORF3b) actively blocks the induction of type I interferon, a crucial aspect of that response.
This protein “is clearly a very good blocker of these early innate defenses of the cells, and it relates perfectly to what we see in organoids and animal models and in COVID-19 patients,” says Benjamin tenOever, a virologist at the Icahn School of Medicine at Mount Sinai in New York. “The virus—for reasons like ORF3b—is very good at shutting off and minimizing the amount of this key antiviral defense called interferon, which plays a really important role in slowing virus infection and inhibiting further replication while you wait for your T cells and your B cells to come in.”
tenOever was not involved in the current study, but in a paper that’s now been published in Cell, which The Scientist reported on as a preprint, his group described how underwhelming the innate immune response to SARS-CoV-2 is. “It’s a bioRxiv paper, so you always have to be a little bit careful that it hasn’t been heavily scrutinized by unbiased reviewers, but, that said, it also makes a lot of sense,” he adds.
Based on clinical reports of COVID-19 and SARS patients, it was apparent to Kei Sato, a virologist at the Institute of Medical Science at the University of Tokyo, and colleagues that the virulence and pathogenicity of SARS-CoV-2 differ from those of SARS-CoV, the coronavirus that caused the SARS outbreak in more than 8,000 people in 2003. “We just hypothesized that the functions of some viral genes are different between them,” Sato writes in an email to The Scientist.
It was unbelievable that a 22–amino acid protein suppresses type I interferon.—Kei Sato, University of Tokyo
To test this idea, the researchers compared the lengths of viral genes between the two coronaviruses. They noticed the SARS-CoV-2 ORF3b is considerably shorter than its SARS-CoV ortholog, encoding a protein just 22 amino acids long, compared to the 154 amino acids of SARS-CoV ORF3b, thanks to the presence of premature stop codons. The team also found that this short ORF3b protein is conserved in SARS-CoV-2’s closest coronavirus relatives, which infect bats and pangolins—scaly-skinned mammals found in Asia and Africa.
Previous work had shown that SARS-CoV ORF3b could inhibit the activation of type I interferon, so the researchers wanted to test whether SARS-CoV-2 ORF3b and related proteins could do the same. They produced a number of ORF3b proteins in human cells and then infected the cells with a mouse respiratory virus that causes a robust immune response in culture. The ORF3b proteins that were best at inhibiting the interferon response were from SARS-CoV-2 and closely related coronaviruses, as well as two proteins from bat viruses related to SARS-CoV that also had shorter ORF3b proteins. When they introduced a premature stop codon into the SARS-CoV ORF3b coding sequence, the resulting 135-amino acid protein was better at suppressing type I interferon than the wildtype SARS-CoV protein was.
“At first, honestly, we just imagined that SARS-CoV-2 ORF3b does not possess the ability to hamper the activation of type I interferon” because of its short length, Sato tells The Scientist. “But the answer was completely opposite to our hypothesis. It was unbelievable that a 22–amino acid protein suppresses type I interferon.”
While it appeared that shorter ORF3b proteins were better at helping the virus duck host immune responses, there wasn’t a linear relationship between length and efficacy. When the team removed the premature stop codons along the length of the SARS-CoV-2 ORF3b sequence to produce proteins 57, 79, 119, and 155 amino acids long in cell culture, all of these except the longest one were better able to suppress the interferon response in cells than the wildtype 22–amino acid ORF3b protein was.
The researchers also determined that at least two COVID-19 patients in Ecuador were infected with a variant of SARS-CoV-2 that encodes a 56-amino-acid-long ORF3b protein. One of the patients died, but Sato cautions “that there is no direct evidence indicating that such quasi-species (encoding the extended ORF3b) are highly pathogenic.”
Studying viral proteins in culture is informative, but it’s not yet clear if blocking ORF3b would help treat the disease, says Priya Luthra, a virologist at the Trudeau Institute in New York who did not participate in the study. Some people with COVID-19 experience a robust, dysregulated innate immune response, she adds, but “the key question is what is really causing this dysregulation in the patients?”
Y. Konno et al., “SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant,” bioRxiv, doi:10.1101/2020.05.11.088179, 2020.