Presence of nucleoprotein makes SARS CoV-2 spike pseudoviruses more infectious

Tarun Mishra and Sreepadmanabh M’s joint interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Tarun and Sreepadmanabh are joint-first authors on the recent research paper “SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles”, published in the journal Frontiers in Cellular and Infection Microbiology (2021). In this interview, they talk about this work and its relevance in the context of the ongoing COVID-19 pandemic.

Tarun Mishra is a Ph.D. scholar at the Dept. of Biological Sciences, IISER Bhopal. Tarun completed his B.Sc from the University of Delhi in 2013. He qualified for the JNU CEEB exam and obtained an M.Sc in Biotechnology from Jammu University in 2016. He cleared the NET and GATE examinations and enrolled at IISER Bhopal as a Ph.D. student in 2017 under the guidance of Dr. Ajit Chande. His research work is mainly focused on the use of lentiviral vectors to study the interactions of emerging viruses with host cellular machinery. Currently, he is also working towards understanding the infectivity profiles of various spike variants derived from the SARS CoV-2.

Sreepadmanabh M is an MS student at the Molecular Virology Laboratory, IISER Bhopal, where he is part of Dr. Ajit Chande’s research group. Currently enrolled in IISER Bhopal’s five-year BS-MS Program, he has previously interned at IISc, won a silver medal at the iGEM (International Genetically Engineered Machines) Competition 2019, and is a KVPY Fellow. With major interests in virology and immunology, he is excited about projects focused on the biology of diseases. Apart from academic pursuits, he is enthusiastic about community-centric initiatives and has had extensive extra-curricular engagements.

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How would you explain your paper’s key results to the non-scientific community?

COVID-19 has rapidly emerged as an unprecedented catastrophe on a global scale. In this regard, the medical and scientific community have been at the forefront of studying and understanding the causative pathogen, the RNA virus termed SARS CoV-2. Since this virus is contagious, it requires highly sophisticated Biosafety Level – 3 (BSL3) facilities to safely work with. Hence, an alternate approach called “pseudotyping” has been adopted to circumvent this issue. Essentially, this involves using the SARS CoV-2’s spike protein (which acts like the virus’ outer envelope) as a layer over a different virus core which is suitably modified to make it non-pathogenic. This approach allows for the production of pseudo-viral particles that carry this spike protein on the surface, thereby mimicking the SARS CoV-2, without the infectious contents of the actual virus. Additionally, custom-designed reporters (e.g. fluorescent protein-encoding genes) may be packaged into these viral particles, which permits a convenient and safer method to observe and study these in more accessible Biosafety Level – 2 laboratories.

Our study picks off from this point. While such pseudotyping methods are a routine and widely accepted standard, we were curious whether including other viral proteins along with the spike would influence the outcomes of these experiments. This was motivated by the fact that in a real-world scenario, the spike protein does not operate in isolation – rather, there are hosts of other viral proteins engaged in various accessory roles during the course of infection.

Figure 1: The top part of the figure shows the experimental setup used to generate spike-enveloped pseudoviral particles. The plasmids required for each specific component have been shown alongside a simplified schematic representation of the protein encoded by each.
The bottom part graphically summarizes this study. When N protein is included during virus preparation, the viral particles produced incorporate a higher amount of spike protein and show increased infectivity. The extent of infectivity is indicated by cells, which express the green fluorescent protein packaged within the viruses.

To address this, we produced such spike-enveloped pseudoviruses in the presence of twenty-four different SARS CoV-2 genes and checked the infectivity of each combination. Eventually, we discovered that the N protein (nucleocapsid protein, an essential component for the assembly of the SARS CoV-2 virus) was substantially enhancing the infectivity of these pseudoviruses. Using multiple molecular and biochemical methods, we established that this effect could be observed even with mutant versions of the spike protein (the popularly termed “UK variant”). Finally, we worked out the likely mechanism by which N protein achieves this. Essentially, N appears to increase the amount of spike protein incorporated into each viral particle, which would make each individual particle more likely to invade cells successfully.

this is one of the first studies to report such a role for the N protein in the context of SARS CoV-2 infectivity, which raises an exciting host of questions for related research in this area.

What are the possible consequences of these findings for your research area?

This work has established that it is important to consider viral factors other than just the spike protein while designing studies aimed at identifying therapeutic drugs, neutralizing antibodies, entry inhibitors, etc. since the actual infectivity levels may vary significantly based on the presence of accessory proteins like N. This could also have implications for the design of viral vector-based vaccines, since including N may help develop more robust immune responses while also improving the overall efficacy and stability of the spike-expressing viral particles.

Indeed, this is one of the first studies to report such a role for the N protein in the context of SARS CoV-2 infectivity, which raises an exciting host of questions for related research in this area. Dissecting the interactions between the spike and N protein would help expand our understanding of SARS CoV-2 pathogenesis and disease biology.

What was the exciting moment (eureka moment) during your research?

Definitely the day we first observed how strongly the N protein enhanced infectivity. Honestly, it was quite a bit of luck. We did a preliminary experiment with twenty-four genes, and while both of us were observing the plate, we were quite fixated on a different gene, which seemed to be modestly increasing the signal. In fact, we were so engrossed with this that we completely missed the part of the plate containing the N protein. And just a while later, Dr. Ajit (who hadn’t visually observed the plate but instead was going over the raw data from the plate reading instrument) rushed in quite excitedly and told us about how impressed he was by N’s effect. It completely blew our minds when we put the plate under the microscope, and there it was, clear as day! The enhancement was so profound, we had to check it thrice over just to convince ourselves that this was for real.

What they say is indeed true – the Devil is in the details!

What do you hope to do next?

We plan to carry forward these results by exploring potential associations of the N protein with spike across different cellular contexts and also including emerging variants of the SARS CoV-2 in related studies.

Where do you seek scientific inspiration?

For this study, well, we literally lived it! Both of us are volunteers at our Institute’s COVID-19 Diagnostic Testing Center, and we are acutely aware of the ground realities of this pandemic. That in itself was sufficient inspiration to leverage the available resources and chip in for the global effort.

In general, our lab has a dedicated culture of discussing and analyzing the latest research in the field and comprehending these advances not just for their importance, but also their scientific merit. Excellence is probably the most potent motivator there is. When you realize that your peers are carrying out such elegant and exceptional work, it imparts a strong drive to make the best use of your opportunities and put in your bit.

How do you intend to help Indian science improve?

A confluence of expertise is important. Large interdisciplinary teams of specialized experts lead a significant proportion of the truly influential and path-breaking papers from the West. While this culture is definitely catching up in India, it yet needs to percolate thoroughly. Collaboration is a crucial ingredient for success in these competitive fields. For instance, we would never have been able to wrap up our study in a few months had we not had access to these viral proteins – which was only possible due to an intra-departmental tie-up. Studies that are more ambitious necessitate such constructive interactions, especially given the abundance of highly specialized and technical tools at our disposal these days. Structural and computational biologists teaming up with more conventional “wet lab” groups make for a very powerful combination. Personally, we intend to actively develop expertise over a wide array of skill sets and specializations over time, such that we would be able to actively contribute towards similar collective efforts.

Reference

Mishra T, Sreepadmanabh M, Ramdas P, Sahu AK, Kumar A, Chande A. SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles. Front Cell Infect Microbiol. 2021;11:663688. doi:10.3389/fcimb.2021.663688

Dr. Ajit Chande lab: https://bio.iiserb.ac.in/faculty_profile.php?id=MTU=&lname=YWppdGc=

Edited by: Anjali Mahilkar

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