Mutation of Nucleocapsid, Not Spike Protein, Responsible for Delta Variant’s Increased Transmissibility

Since the emergence of the Delta variant as the world’s predominant variant of SARS-CoV-2, little has been discovered regarding its mechanism for increased transmissibility. Researchers from the Gladstone Institute of Data Science and Biotechnology (San Francisco, CA) and the Innovative Genomics Institute at UC Berkeley (Berkeley, CA) believe they have found a key site of genetic mutations in the nucleocapsid that could be responsible for the Delta variant’s increased transmissibility.

One author of the study, Abdullah Syed, described in a press release that the life cycle of a virus can be divided into three parts:

• Entry: the virus is enters a cell
• Replication: the virus hijacks the cell, causing it to create more copies of proteins and genetic material that comprise the virus
• Assembly: the copied proteins and genetic material are packaged into new virus particles

The nucleocapsid, a multifunctional structural protein, is critical to the efficiency of the assembly stage in the coronavirus. Though it was previously hypothesized that mutations in the spike protein were causing increased efficiency during the entry stage, the researchers found that mutations in the nucleocapsid were the most significant contributor to Delta’s higher infectivity.

Typically, researching the nucleocapsid would require real viruses, because unlike the spike protein, it is located inside of the virus. This has caused nucleocapsid research to be overlooked, as working with real viruses is inherently dangerous due to the possibility of infecting researchers.

Taha Taha of the Gladstone Institute of Virology described the method that enabled the researchers to study virus replication without using live SARS-CoV-2 virions. They used virus-like particles (VLPs), which have the same structure as the virus, but lack any genetic material for replication. This means that researchers do not risk infection, as the VLPs are unable to replicate. VLPs are also easier to mutate than live viruses.

The researchers genetically engineered the VLPs to express luciferase (the enzyme that causes fireflies to glow), as the light it gives off can be used to gauge activity of a protein-expressing gene. By mutating the nucleocapsid with the mutations found in the Delta variant, an increase in luciferase expression (measured by light) would signal that the mutations increase the functionality of the nucleocapsid.

This was confirmed as one single amino acid mutation (the most basic mutation possible) in the nucleocapsid was found to cause a tenfold increase of luciferase expression, meaning that the mutated virus-assembly protein was ten times as active. Syed noted that this matched the tenfold increase in viral load observed in patients infected with the Delta variant.

The researchers further proved that the mutations increased the activity of the nucleocapsid by infecting cells with real SARS-CoV-2 virions in a highly controlled lab setting, finding that the real mutated virus also demonstrated faster reproduction.

This research has the potential to completely redirect the focus of scientists searching for culprit mutations in new variants of SARS-CoV-2, as research has largely focused on the spike protein until now. An improved understanding of the mechanisms of improved infectivity is important to researchers developing new therapies.

The novel virus-like particle method used could also prove to change virus research forever, as Taha states that this faster and safer alternative to using real viruses could also be used to test existing therapeutics (like vaccines) on new variants. Syed mentioned that the VLP method could even be used to find out if certain animal coronaviruses are capable of infecting humans, to develop new methods of therapy for COVID-19, or to do research on newly emerging viruses that are potentially too dangerous to work with.