Understanding the mechanism of SARS-CoV-2 replication has become extremely vital so as to obtain possible antiviral strategies to suppress the intensity of the deadly pandemic.
Coronaviruses are of four types: alpha-, beta-, gamma- and delta-coronavirus. Betacoronaviruses are responsible for Severe Acute Respiratory Syndrome(SARS), and Middle-east Respiratory Syndrome (MERS) outbreaks during the early 2000s as well as the COVID-19 pandemic. These viruses can cause severe pneumonia and bring about lethal complications in humans. Being zoonotic in nature, coronaviruses infect animals from which they spread to humans. The exact zoonotic pathway for SARS-CoV-2 is unknown even though it is established to have originated from bats. The infective ability of these zoonotic viruses depends on how they enter into the host cell via surface ACE-2 (angiotensin-converting enzyme-2) receptors, evade the immune response and use the host machinery to make more copies of themselves.
An international collaborative study led by Prof. Albrecht Von Brunn of Ludwig Maximillian University, Germany, went on to study what are the strategies employed by the coronavirus to create more arsenal for infection. These findings were published in The EMBO Journal. Key findings include the involvement of a highly conserved SARS-Unique Domain (SUD) on the protein sequence of Non-structural protein-3 (Nsp-3) of the SARS-CoV-2 in forming newer viral particles.
The structure and function of the SUD domain:
The amino acid sequence identity of SARS-CoV and SARS-CoV-2 proteins share 75% similarity. This SUD region is found on the Nsp-3 proteins of both viruses. This domain is found to interact with poly-A binding protein (PABP), PABP interacting protein-1 (Paip-1) and ribosomes, all of which are involved in the translation process (formation of proteins from mRNA).
The SUD domain has three subdomains: the N terminal, the middle part and the C-terminal. It is the N terminal macrodomain that interacts with the host proteins and initiation factors involved in translation initiation. These SUD-like domains were also observed in the coronaviruses that infect bats. The N terminal and middle part of the SUD region bind to guanine and adenine oligomers in the host genome to form a tight complex as well as to other proteins which can degrade the antiviral p53 protein, thus enhancing viral replication.
During normal conditions, Paip-1 protein positively stimulates host translation initiation by enhancing the activity of PABP protein which binds to the poly A tail found on the 3’ end of the mRNA. Paip-1 also interacts with several initiation factors so as to ensure that only intact mRNA gets converted to protein hence stimulating efficient translation.
The study validated many molecular associations between the SUD domain and host Paip-1 protein via size exclusion chromatography, split-yellow protein fluorescence, and co-immunoprecipitation assays. The N terminal of the SUD domain links to the middle domain of the Paip-1 proteins from the lysine residue on the 389th position to the threonine residue on the 404th position. This interaction can be a possible antiviral target. SUD region and PABP protein do not compete with each other to bind to Paip-1 protein instead they form a ternary complex. SUD also enhances the binding between PABP and Paip-1 (4.4 fold increase). It also associates with the 80S ribosome complex through the 40S ribosomal subunit which helps in holding the viral mRNA in place. Through these mechanisms, SUD increases the rate of viral protein production.
The next puzzle to solve was whether the SUD domain enhances host protein production or viral protein formation. The researchers created a mimetic of an infected SARS-CoV-2 cell. Through luciferase activity assay, it was observed that SUD selectively enhances viral protein translation and halts the host protein formation. Parts of the Nsp-1 region other than the SUD domain also go on to degrade the host mRNA as well as block it from binding to the ribosome thereby preventing any more host proteins to be produced.
The mRNA of the coronavirus forms a closed-loop structure which blocks the host mRNA from binding to the ribosome. The SUD region along with the proteins involved in translation organises into an initiation complex with the ribosome that attracts the initiation factors increasing the rate of translation of the viral proteins. The viral proteins are essential for the assembly of newer virus particles.
RNA viruses always seek to invade the host machinery and utilise them to its benefit. Coronaviruses adopt unique strategies making it challenging to find out ways to suppress their infective activity. “More structural information on virus:host protein complexes in this field is necessary to arrive at a better understanding,” assert the researchers.