Unraveling the Enigma of SARS-CoV-2’s Cryptic ORF3c: An Immune Evasion Factor


Since the emergence of the COVID-19 pandemic, extensive research efforts have been directed toward unraveling the mysteries surrounding the SARS-CoV-2 virus. While canonical proteins of the virus have been well-characterized, the existence of smaller open reading frames (ORFs) that overlap with other ORFs has sparked curiosity within the scientific community.

These cryptic ORFs may play crucial roles in viral replication, immune evasion, and pathogenicity, and they hold the key to understanding some of the unique characteristics of SARS-CoV-2.

One such cryptic ORF is ORF3c, nestled within the SARS-CoV-2 genome, with its specific location identified as nt 25457-25579 of the Wuhan-Hu-1 reference genome. Its existence has been independently reported by various research groups, resulting in several alternative names such as ORF3h, 3a.iORF1, and ORF3b. In this article, we will use the term ORF3c to refer to this intriguing open reading frame. Unlike canonical genes, the functions and implications of these cryptic ORFs have remained enigmatic, raising questions about their role in the virus’s biology.

This article delves into the latest research findings that shed light on ORF3c and its potential significance in the SARS-CoV-2 virus. It provides an in-depth analysis of its structure, expression, and function, highlighting its role as an immune evasion factor.

Cryptic ORFs: An Overlooked Aspect of SARS-CoV-2

SARS-CoV-2 is known for its canonical proteins, which play crucial roles in viral replication and host interaction. However, several smaller open reading frames (ORFs) have received limited attention until recently. These cryptic ORFs often overlap with the canonical genes, raising questions about their potential functions and impact on the virus’s biology.

One such cryptic ORF that has come under scrutiny is ORF3c. This particular ORF, residing in close proximity to the well-known ORF3a, has garnered attention from researchers for its possible involvement in SARS-CoV-2’s immune evasion strategies. Despite being cryptic and poorly understood, ORF3c has now emerged as a key player in modulating the host’s immune response.

The Enigmatic ORF3c: Structure and Function

ORF3c, initially referred to by different names such as ORF3h and 3a.iORF1, is located in the SARS-CoV-2 genome at nt 25457-25579. Early in silico analyses indicated that the ORF3c protein may possess a transmembrane domain, suggesting its role as a viroporin. However, until recently, the true nature of ORF3c and its relevance to viral replication remained unclear.

Recent research efforts have shed light on the structure and function of ORF3c. It has been revealed that ORF3c encodes a stable 41-amino-acid peptide that is expressed in virally infected cells. What sets this cryptic ORF apart is its ability to suppress the induction of IFN-β expression, a vital component of the host’s antiviral response.

Mechanistic studies have unveiled that ORF3c inhibits the innate sensing triggered by RIG-I and MDA5 and interacts with MAVS, an essential adaptor protein in the host’s immune response.

ORF3c’s suppression of RIG-I’s interaction with MAVS and its induction of proteolytic cleavage of MAVS further emphasize its immunosuppressive activity. Importantly, this activity is not unique to SARS-CoV-2; ORF3c orthologs from various sarbecoviruses share this capacity, highlighting the evolutionary importance of this cryptic ORF.

Evolution and Variation: Cryptic ORF3c Across SARS-CoV-2 Lineages

The existence of ORF3c and its immunosuppressive function has raised questions about its prevalence and necessity within different SARS-CoV-2 lineages. Intriguingly, despite its ability to suppress the host immune response, some SARS-CoV-2 lineages exhibit premature stop codons within their ORF3c genes. This finding suggests that ORF3c is not universally essential for the virus’s survival and spread within the human population.

Research has also explored the impact of disrupting ORF3c on viral replication in various cell types, including CaCo-2, CaLu-3 cells, and bat lung cells. Surprisingly, the results indicate that the absence of ORF3c does not significantly affect SARS-CoV-2 replication. This finding suggests that while ORF3c plays a critical role in immune evasion, it is not a crucial determinant of the virus’s replicative success.


The discovery of the ORF3c gene in SARS-CoV-2 and its potential involvement in viral replication has presented an intriguing puzzle for researchers. Various lines of evidence have hinted at the importance of this cryptic ORF in the virus’s life cycle. This discussion delves into the implications of the findings, addresses the role of ORF3c as an immune evasion factor, and explores its significance within the context of viral evolution.

A Key Player in Viral Replication

The initial clues that ORF3c might play a role in viral replication were gleaned from multiple sources. These included the conservation of the open reading frame in different sarbecoviruses, synonymous site conservation, and the presence of upstream ATGs that may facilitate ORF3c translation through leaky scanning. Ribosomal profiling and HLA-II immunopeptidome data further supported the hypothesis that ORF3c is actively translated in SARS-CoV-2-infected cells.

Additionally, ORF3c exhibited a high density of CD8+ T-cell epitopes, indicating its potential as a functional protein. In silico analyses predicted a transmembrane domain in ORF3c, hinting at its potential role as a viroporin. Despite these clues, the actual expression of ORF3c in infected cells, its precise function, and its contribution to efficient viral replication had remained unclear.

ORF3c as an Immune Evasion Factor

The latest research unequivocally identifies ORF3c as an immune evasion factor, not only for SARS-CoV-2 but also for other sarbecoviruses. ORF3c inhibits the induction of IFN-β by impeding the activation of innate sensing cascades, specifically through RIG-I and MDA5 pathways. The generation of a replication-competent SARS-CoV-2 variant encoding a tagged ORF3c confirmed the presence of a stable ORF3c peptide in virus-infected cells. Luciferase reporter assays demonstrated that ORF3c significantly suppresses the activation of the IFNB1 promoter, revealing its inhibitory effect on the host’s antiviral response.

Furthermore, experiments involving Sendai virus, a known RIG-I sensor, underscored ORF3c’s ability to reduce the expression of endogenous IFN-β. This suggests that ORF3c intervenes at a point downstream in the signaling cascade, implicating an interaction with the mitochondrial signaling adaptor MAVS. This interaction was confirmed through co-immunoprecipitation experiments. Remarkably, ORF3c appears to prevent RIG-I’s interaction with MAVS and induces the proteolytic cleavage of MAVS, potentially by removing its C-terminal transmembrane domain.

ORF3c’s Conservation and Variations in Sarbecoviruses

The immunosuppressive activity of ORF3c is not limited to SARS-CoV-2 but extends to orthologs in other sarbecoviruses, including the SARS-CoV reference virus Tor2. However, the activity and expression levels of these orthologs may vary due to single amino acid changes. For instance, the L11P mutation in ORF3c distinguishes it from the highly active Wuhan-Hu-1 ORF3c. Polymorphisms at specific positions within ORF3c, such as Leu2/Leu3 and Ile6/Leu7, contribute to the immunosuppressive effect and its interaction with MAVS. These residues are largely conserved among sarbecoviruses, with one exception being Ile6, which is replaced by valine in SARS-CoV variants.

ORF3c in the Context of Other Immune Evasion Factors

ORF3c joins a growing list of SARS-CoV-2 proteins that interfere with RIG-I- and/or MDA-5-mediated immune activation. This includes ORF10, which targets MAVS for inhibition, and other proteins like ORF3b, nucleocapsid, ORF6, and ORF8 that have been shown to suppress IFN-β expression. The presence of multiple proteins with similar immune evasion activities suggests a backup mechanism that ensures viral replication, even if one of the IFN-β-suppressing proteins is lost.

Moreover, recent studies have unveiled small open reading frames within SARS-CoV-2’s negative-sense RNA, referred to as rORFs, which also suppress IFN-β promoter activity. The redundancy in viral immune evasion mechanisms emphasizes the critical role of escaping the host’s antiviral response in the virus’s survival strategy.

The Enigma of ORF3c in Viral Replication

Despite the discovery of ORF3c as an immune evasion factor, questions about its relevance in viral replication persist. It’s evident that ORF3c is not an essential determinant of efficient viral replication in vitro or in vivo, as the loss of ORF3c does not significantly impact viral replication or IFN-β expression in infected cell lines.

The presence of premature stop codons in the ORF3c gene of certain SARS-CoV-2 variants has not hindered their efficient spread in the human population. This observation further emphasizes the idea that ORF3c may not be a decisive factor for viral replication. It is possible that other viral proteins, such as ORF8 or nucleocapsid, can compensate for the loss of ORF3c.

Future Directions and Unanswered Questions

The investigation into ORF3c and its potential role in viral replication is far from over. Further experiments in animal models will provide insights into the impact of ORF3c on immune activation and viral replication within an infected organism. The dynamics of ORF3c variants, co-evolution with other viral genes, and potential associations with viral loads, disease progression, or transmissibility will be vital areas of research.

The emergence of a nucleotide change in a subset of B.1.617.2 viruses, reverting their ORF3c stop codon, underscores the complexity of viral evolution. It suggests that the loss of ORF3c was initially carried along with mutations elsewhere in the genome that conferred a significant fitness advantage to the virus before beneficial ORF3c expression was restored by another point mutation.

In summary, ORF3c represents an intriguing piece of the SARS-CoV-2 puzzle. While it has been identified as an immune evasion factor, its direct impact on viral replication and its co-evolution with other viral genes remain open questions. The continued study of ORF3c and its variants, both in vitro and in vivo, will undoubtedly provide more answers and insights into the complex world of SARS-CoV-2 and its interactions with the host’s immune system.


In conclusion, the study of cryptic ORFs, such as ORF3c, within the SARS-CoV-2 genome continues to unveil exciting insights into the virus’s biology and immune evasion strategies. While many questions about these cryptic ORFs remain, recent research has illuminated the structure, expression, and function of ORF3c, confirming its role as an immune evasion factor.

ORF3c’s ability to suppress the host’s antiviral response by inhibiting RIG-I and MDA5 and interacting with MAVS highlights its significance in the virus’s survival strategy. Moreover, the presence of ORF3c orthologs in different sarbecoviruses underscores its evolutionary importance.

However, the existence of SARS-CoV-2 lineages with disrupted ORF3c genes and the minimal impact of ORF3c disruption on viral replication suggest that while this cryptic ORF is a key player in immune evasion, it is not an essential component of efficient viral replication.

The ongoing study of cryptic ORFs within SARS-CoV-2 promises to unravel more secrets about this enigmatic virus, potentially leading to new insights that can inform therapeutic strategies and our understanding of viral pathogenesis.

reference link : https://www.embopress.org/doi/full/10.15252/embr.202357137


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