Unveiling the Molecular Mechanisms of SARS-CoV-2 Pathogenesis: The Role of CoV-2 Nsp13 and CoV-2 N Proteins

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Since December 2019, the world has been grappling with the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has had far-reaching consequences for both the global economy and individual health.

SARS-CoV-2 is primarily an airborne virus, and its mode of transmission primarily occurs through oral and nasal inhalation. The clinical symptoms of SARS-CoV-2 infection range from mild symptoms like headache, cough, fever, sore throat, fatigue, myalgia, and dyspnea to severe cases that may result in death.

In comparison to previous coronaviruses like severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 is highly infectious, making it a significant global health threat.

As of July 2021, it had infected over 195 million people and caused over 4.2 million deaths worldwide, underscoring the urgent need to understand the molecular mechanisms underlying SARS-CoV-2 pathogenesis, immune evasion, and disease progression.

The Structure of SARS-CoV-2

SARS-CoV-2 belongs to the beta coronavirus family within the Coronaviridae family. It is a spherical, encapsulated, positive-sense single-stranded RNA virus with a genome length of approximately 30 kb. The viral genome contains 11 genes that encode about 20 functional proteins.

These genes include the 5′ untranslated region, replicase polysaccharide protein gene (ORF1/ab), spike glycoprotein gene (S), envelope glycoprotein gene (E), membrane glycoprotein gene (M), nucleocapsid protein gene (N), and the 3′ untranslated region.

Importantly, SARS-CoV-2 exhibits several highly conserved sequences among different mutants and other coronaviruses, with particular emphasis on the nonstructural protein 13 (Nsp13) and the nucleocapsid protein (N). Understanding the structure and function of these proteins is crucial for the development of anti-SARS-CoV-2 drugs.

The Role of CoV-2 Nsp13

CoV-2 Nsp13, a protein composed of 596 amino acids, plays a significant role in SARS-CoV-2 replication. Its structure consists of five domains: two RecA-like helicase subdomains (1A and 2A) forming the triangular base, an N-terminal zinc-binding domain, a helical “stalk” domain, and a beta-barrel 1B domain. CoV-2 Nsp13 acts as a helicase, unwinding duplex RNA, and a 5′-triphosphatase, likely involved in capping viral mRNA.

Moreover, it strongly inhibits type I interferon signaling, which is essential for antiviral immune responses. Recent studies also suggest that CoV-2 Nsp13 is involved in blocking immune activation during infection, particularly in blocking interferon (IFN) and NF-κB activation. This multifunctional protein even exhibits 5′-3′ unwinding activity on double-stranded DNA and RNA, further highlighting its versatility during viral infection.

The Role of CoV-2 N

CoV-2 N, composed of 413 amino acid residues, is the sole protein that binds to genomic RNA in the nucleocapsid. It plays a pivotal role in viral replication and regulation of cell signaling pathways. Notably, CoV-2 N is among the most conserved proteins in coronaviruses. Its high immunogenicity makes it a potential candidate for vaccines against various human coronavirus strains. Some studies have proposed it as a crucial target for combating COVID-19, emphasizing the importance of understanding its biochemical functions.

CoV-2 N as an RNA and DNA Binding Protein

Initially, CoV-2 N was recognized as an RNA-binding protein essential for viral genome packaging. However, recent research has revealed that it can also bind to host mRNAs, potentially interfering with normal host cell functions. Importantly, CoV-2 N can bind to DNA nonspecifically, raising questions about its functions in host cells. This characteristic is similar to the N protein of the human immunodeficiency virus (HIV), which also binds to DNA to perform various cellular functions.

Comparing CoV-2 Nsp13 and CoV-2 N

While CoV-2 Nsp13 is primarily known for its helicase activity, CoV-2 N, despite its structural role, can also open double-stranded nucleic acids. The significant sequence and structural differences between these two proteins suggest that their mechanisms for opening nucleic acid substrates differ.

This unique biochemical characteristic of CoV-2 N is akin to that of single-stranded binding proteins found in both prokaryotes and eukaryotes, like Escherichia coli single-stranded DNA-binding protein (SSB) and replication protein A (RPA), respectively, both of which exhibit double-stranded DNA unwinding activity.

Discussion

CoV-2 N and CoV-2 Nsp13: Crucial Proteins in SARS-CoV-2 Replication

In the quest to understand the molecular intricacies of SARS-CoV-2 and develop potential therapeutic strategies, two proteins have emerged as central players: CoV-2 N and CoV-2 Nsp13. These proteins play indispensable roles in viral replication, and their functions are highly conserved among coronaviruses.

CoV-2 N is known for binding to genomic RNA in the nucleocapsid, while CoV-2 Nsp13 exhibits the highest sequence conservation among coronaviruses, emphasizing its importance in viral viability. Consequently, CoV-2 Nsp13 represents a promising target for the development of antiviral drugs, further underscoring the need for a systematic analysis of the biochemical characteristics of both CoV-2 N and CoV-2 Nsp13 in vitro.

Diverging Functions: CoV-2 N and CoV-2 Nsp13

CoV-2 Nsp13 and CoV-2 N, despite their relatedness in the viral life cycle, exhibit distinct primary sequences and structural properties. CoV-2 Nsp13 contains structurally conserved domains characteristic of typical helicases, including ATP-binding and ATP-hydrolysis sites, distinguishing it as a true helicase.

Conversely, CoV-2 N does not possess any domain with a predicted unwinding function, making it quite different from CoV-2 Nsp13 in this regard. This implies that CoV-2 N, resembling a single-strand binding protein, opens duplexes through a mechanism distinct from typical helicases.

In Vitro Analysis: Unwinding Activities of CoV-2 N and CoV-2 Nsp13

To investigate the unwinding activities of CoV-2 N and CoV-2 Nsp13, an in vitro gel assay system was established, marking the first-time researchers systematically studied these activities. Notably, while CoV-2 Nsp13 exhibits unwinding activity, no prior reports have indicated such activity for CoV-2 N or other coronavirus nucleocapsid proteins. The two proteins are indeed quite dissimilar in their mechanisms, which raises important questions about their functions.

CoV-2 N Unwinding Characteristics

CoV-2 N unwinds dsDNA under specific conditions. Importantly, CoV-2 N does not require the typical cofactors like Mg2+, DTT, or NTP as helicases do. Instead, it unwinds dsDNA effectively in the presence of 100–300 mM NaCl. This is in contrast to CoV-2 Nsp13, which demonstrates superior unwinding activity at low NaCl concentrations. It’s worth noting that high NaCl concentrations (>400 mM) inhibit CoV-2 N binding to ssDNA, suggesting that the unwinding ability weakens under such conditions. This observation underscores the unique unwinding characteristics of CoV-2 N.

Furthermore, CoV-2 N’s unwinding activity is distinct from that of typical single-strand binding proteins like RPA. RPA requires specific temperature conditions for unwinding, while CoV-2 N unwound dsDNA at 25°C and 30°C. This indicates that CoV-2 N may not conform to the traditional behavior of single-strand binding proteins.

CoV-2 Nsp13 Unwinding Activity

In contrast, CoV-2 Nsp13, the sole protein with an unwinding domain in the SARS-CoV-2 genome, closely resembles typical helicases in its unwinding properties. It exhibits unwinding activity when the single-stranded bubble structure is at least 12 nucleotides in length, with activity increasing as the tail length grows.

CoV-2 N and CoV-2 Nsp13 Annealing Activities

The findings also revealed differences in the annealing activities of CoV-2 N and CoV-2 Nsp13 at equivalent and increasing concentrations. CoV-2 N demonstrated an unusual shift between annealing and unwinding activities with changing concentrations. At low concentrations, annealing was dominant, whereas unwinding became prominent at higher concentrations. This suggests that CoV-2 N can regulate the transition between these activities based on its concentration.

Implications and Future Research

The role of CoV-2 N’s unwinding function in viral and host cell processes remains enigmatic and merits further investigation. An intriguing hypothesis relates to the subcellular localization of the N protein within host cells. In the cytoplasm, it participates in viral genome replication, transcription, and packaging, primarily through its RNA-binding and -annealing activities. However, in the nucleus, the N protein plays a role in cell cycle regulation, which may affect host cell replication and viral packaging.

In conclusion, the unraveling of the unwinding activities of CoV-2 N and CoV-2 Nsp13 in this study sheds light on the multifaceted functions of these proteins in the viral replication cycle. These discoveries open new avenues for understanding SARS-CoV-2 biology and may have implications for future therapeutic strategies. Further research will be essential to decipher the precise roles and regulation of these proteins in the context of viral infection and host cell interactions.

Conclusion

The SARS-CoV-2 pandemic has highlighted the urgent need to explore the molecular mechanisms of viral pathogenesis and develop strategies for combating the virus. Key proteins like CoV-2 Nsp13 and CoV-2 N play crucial roles in viral replication, immune evasion, and host cell interactions. Understanding their functions and biochemical characteristics can offer valuable insights for drug development and vaccine research. As research continues, further revelations about these proteins may provide the key to effectively combating the ongoing pandemic and preparing for future viral threats.


reference link : https://www.frontiersin.org/articles/10.3389/fmicb.2022.851202/full

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