The Hidden Depths of Viral Genomes: Unraveling the Complexities of +ssRNA Viruses

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Viruses, often regarded as the epitome of biological simplicity, are submicroscopic obligate intracellular parasites with typically small genomes. Their classification hinges on the nature of their genomic nucleic acid and replicative intermediates, segregating them into seven distinct classes.

These include the single-stranded RNA viruses (both positive-sense [+ssRNA] and negative-sense [−ssRNA]), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and double-stranded DNA (dsDNA) viruses, along with retroviruses and pararetroviruses. The International Committee on Taxonomy of Viruses provides a comprehensive taxonomy of these classes (https://talk.ictvonline.org/taxonomy/).

Among these, +ssRNA viruses represent a significant portion. They exhibit a range of genomic architectures, from monopartite to tripartite ssRNA genomes. These viruses are notorious for their impacts on both human and animal health, as well as on agriculture. Notable pathogens in this class include the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic, and others like dengue, Zika, and yellow fever viruses. These infections often result in severe economic losses and catastrophic mortality rates.

On the other end of the spectrum are the ssDNA viruses, exemplified by their minimalistic genetic makeup, spanning only about 2 to 6 kilobases. Geminiviruses and nanoviruses, belonging to the Geminiviridae and Nanoviridae families respectively, are prime examples of plant-infecting ssDNA viruses causing significant economic damage.

Viruses, constrained by their small genomes, have evolved ingenious strategies to exploit host cellular mechanisms for their replication and propagation. They employ a variety of noncanonical translation strategies, such as internal ribosome entry and ribosomal frameshifting, to maximize the utility of their limited genetic information. This sophistication is further illustrated by the recent discoveries of “hidden” protein-coding open reading frames (ORFs) within viral genomes, employing these unconventional translation methods.

A groundbreaking revelation in this field was made by Gong et al., who uncovered that +ssRNA viruses encode functional proteins on their (−)-strand replication intermediates (−RNA), challenging the long-held belief that −RNA is merely a replication intermediate devoid of coding potential. This discovery, as published in their study, indicates that these −RNAs contain multiple conserved small reverse ORFs (rORFs), encoding peptides with significant biological functions.


TABLE 1

The revelation by Gong et al. marks a significant turning point in our understanding of viral genetics, particularly regarding +ssRNA (positive-sense single-stranded RNA) viruses. To fully appreciate the magnitude of this discovery, it’s essential to break down the key components of the statement:

Understanding +ssRNA Viruses: These viruses have genomes made of RNA that is in the same orientation as the host’s mRNA, allowing direct use of the viral RNA for protein synthesis by the host’s ribosomes. Common examples include viruses like SARS-CoV-2.

The Concept of (−)-Strand Replication Intermediates (−RNA): In the life cycle of +ssRNA viruses, a critical step is the synthesis of a complementary (−)-strand RNA. This −RNA strand serves as a template for the production of new +RNA strands, which can then be packaged into new virus particles or translated into viral proteins. Traditionally, this −RNA was considered a mere intermediate in replication, not thought to be directly involved in encoding proteins.

The Groundbreaking Discovery: Gong et al. challenged this traditional view by demonstrating that the −RNA of +ssRNA viruses is not just a replication template. Instead, they found that this strand also encodes functional proteins. This means that the −RNA contains sequences (open reading frames or ORFs) that can be translated into proteins, just like the +RNA strand.

Implications of Encoding Proteins on −RNA: The ability of the −RNA strand to encode proteins has several important implications:

  • Complexity of Viral Genomes: This discovery reveals a new layer of complexity in viral genomes, suggesting that they have more coding capacity than previously understood.
  • Viral Evolution and Adaptation: The encoding of proteins on the −RNA strand may be a strategy evolved by viruses to maximize their genetic potential and adaptability, allowing them to produce a wider range of proteins from a limited genome.
  • Challenging Established Dogmas: It overturns a long-held belief in molecular biology that −RNA strands in these viruses are non-coding, prompting a re-evaluation of how we interpret viral genomes.
  • New Avenues for Research and Treatment: Understanding that −RNA strands can encode proteins opens up new avenues for research into viral life cycles and pathogenesis. It may lead to the identification of new targets for antiviral drugs or vaccines.

Technical Challenges and Future Research: This discovery likely required advanced techniques in molecular biology, such as next-generation sequencing and bioinformatics, to identify these hidden ORFs. Future research will need to explore how widespread this phenomenon is across different +ssRNA viruses, the exact functions of the proteins encoded by the −RNA, and how these proteins contribute to the virus’s life cycle and virulence.

In summary, the work of Gong et al. is a paradigm-shifting discovery that challenges traditional views of RNA virus biology, suggesting that the genomes of +ssRNA viruses are more versatile and complex than previously thought, with significant implications for virology and the broader field of molecular biology.


These rORFs, though short and encoding small peptides, are integral to the viral lifecycle. For instance, in SARS-CoV-2, these peptides play a role in suppressing type-I interferon production, aiding in infection efficiency. In the realm of plant viruses, such as those in the Potyviridae family, these peptides are essential for viral pathogenesis. The rORF2 protein of the turnip mosaic virus (TuMV), a member of this family, exemplifies this. It interacts with the viral RNA-dependent RNA polymerase and is crucial for the virus’s survival.

The implications of these findings are profound. They not only challenge our understanding of viral genomics but also open new avenues for antiviral research. The existence of these hidden proteins and their functional roles in viral infection cycles present potential targets for therapeutic interventions.

Most eukaryotic mRNAs are translated in a cap-dependent manner, but as highlighted earlier, viruses, including +ssRNA viruses like SARS-CoV-2 and TuMV, have developed multiple noncanonical mechanisms for efficient translation of viral proteins. These diverse strategies are tailored to meet their specific needs. For instance, Gong and colleagues identified the use of internal ribosome entry sites (IRES) by these viruses to recruit ribosomes, facilitating the translation of proteins encoded by previously overlooked rORFs.

The exploration of +ssRNA viruses extends to narnaviruses, which infect a wide range of organisms. Recent studies have unearthed a large ORF in the −RNA of different narnaviruses, indicating a previously unknown translation mechanism. Dinan and colleagues conducted a comprehensive analysis of narnaviral sequences available publicly, discovering that long rORFs are common in one clade of narnaviruses, sometimes occupying over 95% of the genome. These ORFs are among the longest-known overlapping genes, shedding light on the evolution of overlapping genes and the genesis of new genes.

Furthering this exploration, Zhang and colleagues focused on a novel narnavirus, Puccinia striiformis virus 5 (PsV5), from the wheat stripe rust fungus. They identified a conserved rORF within the −RNA of PsV5 and found that its overexpression increased susceptibility to wheat stripe rust and enhanced Fusarium graminearum virulence. This discovery underscores the biological significance of these rORFs and calls for a reevaluation of the +ssRNA virus proteome, challenging the previous belief that these viruses only encode proteins on the +RNA strand.

Turning our attention to +ssDNA viruses, similar revelations have come to light. Geminiviruses, which are phytopathogenic DNA viruses with circular ssDNA genomes, were previously thought to encode 6 to 8 canonical viral proteins. However, recent studies have uncovered the existence of small hidden ORFs with vital biological functions in these viruses. Gong and colleagues, by redefining the criteria for protein identification, identified several such ORFs in geminiviruses, including V3, C5, and C7, and revealed their specific cellular localizations and roles in virulence. For instance, the C5 protein of tomato yellow leaf curl virus (TYLCV) is shown to anchor to plasmodesmata in infected cells, establishing a functional connection between plasmodesmata and geminivirus cell-to-cell movement. Concurrently, Chiu and colleagues identified hidden ORFs in tomato yellow leaf curl Thailand virus, demonstrating the necessity of translating different protein isoforms for viral pathogenicity in tomato plants.

These findings collectively suggest that both +ssRNA and +ssDNA viruses harbor more complex genetic architectures than previously understood. The discovery of hidden proteins encoded within these viruses not only challenges our current understanding of viral genetics but also opens new avenues for research and therapeutic development. It becomes evident that the viral world is much more intricate than we once believed, necessitating a deeper investigation into the hidden facets of viral genomes to fully comprehend their biology and pathogenicity.


Conclusion: Unveiling the Hidden Layers of Viral Complexity

The recent advancements in virology have significantly altered our understanding of viral genetics and pathogenesis. These revelations about hidden proteins in both +ssRNA and +ssDNA viruses represent a paradigm shift in how we perceive viral genomes and their capabilities. The discovery of novel open reading frames (ORFs) in the −RNA of +ssRNA viruses like SARS-CoV-2, TuMV, and various narnaviruses, and in the genomes of +ssDNA viruses such as geminiviruses, challenges the traditional view of viral coding potential and genome organization.

These findings underscore the remarkable adaptability and complexity of viruses. They have evolved sophisticated mechanisms to maximize their genetic coding capacity, often in ways that defy the conventional understanding of molecular biology. The identification of noncanonical translation mechanisms and hidden proteins not only enriches our understanding of viral replication and pathogenesis but also provides new targets for antiviral strategies.

The implications of these discoveries extend beyond the realm of virology. They have the potential to influence the broader fields of genetics, molecular biology, and bioinformatics. The fact that viruses can encode functional proteins in what was previously considered non-coding regions calls for a reevaluation of genomic analyses across all forms of life. It suggests that what we currently know about genomes, even those well studied, might only be the tip of the iceberg.

Furthermore, these insights have significant implications for public health and agriculture. Understanding the full range of proteins that viruses can produce allows for the development of more effective vaccines and antiviral therapies. In agriculture, this knowledge can lead to the creation of more resistant crop varieties, potentially reducing the impact of viral diseases on food security.

In conclusion, the exploration into the hidden depths of viral genomes is a testament to the ever-evolving nature of scientific discovery. It challenges researchers to look beyond established norms and delve deeper into the unknown. As we continue to unravel the complexities of viral genomes, we may find more surprises that could revolutionize our approach to dealing with viruses, ultimately benefiting both human health and global agriculture. This journey into the microscopic world of viruses reaffirms the notion that in science, there is always more to discover, more to understand, and more to apply for the betterment of humanity.


reference link : https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011905

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