Molecular analysis of human norovirus VP2 elucidates critical molecular interactions for virus assembly

 The article can be read here, and more about the primary investigator can be found here.  

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            Norovirus is one of the leading causes of gastrointestinal illness around the world. Outbreaks are common in schools, nursing homes, and other crowded places and typically stem from contaminated food. As this paper highlights, there are estimated to be 680 million cases per year. Despite the high burden of disease, there is not currently a vaccine or a specific antiviral treatment for norovirus.

One of the main knowledge gaps holding back research into treatments and vaccines is a lack of understanding of the norovirus lifecycle, including how the viral proteins come together to make a new infectious particle. In this study, the authors identify and investigate a crucial component of this process.

 

The building blocks of the norovirus capsid

            The norovirus capsid, or protein shell surrounding the genome, is made of two proteins. The bulk of the capsid is formed from major capsid protein VP1. Compared with the rest of the virus, VP1 is well understood. Its structure can be studied through x-ray crystallography and cryo-electron microscopy, allowing researchers to visualize the protein. In contrast, the minor capsid protein VP2 has not yet been resolved through these methods. While prior research has shown that VP2 and VP1 interact with each other, the structure of VP2 can only be visualized through predictive software like AlphaFold and its function remains largely unknown. This study attempts to answer some of these questions surrounding VP2.

            A third component of this system is VPg, a protein that attaches to the norovirus genome. VPg is crucial for norovirus success, and without it the virus can neither infect cells nor make more virus once inside. VPg is also thought to be part of encapsidation, the process of packaging the viral genome into the capsid after the capsid has been built. To escort the genome inside the capsid, VPg might interact with either VP1 or VP2. As part of their attempt to understand VP2, the authors also determine the nature of its interactions with VPg.

 

Figure 2B: A model generated using the protein folding program AlphaFold shows the predicted shape and structure of norovirus protein VP2.

            The first thing the authors do is figure out if VP2 is different between genogroups of norovirus, and what they find is that VP2 is highly diverse. Most of the protein looks considerably different between norovirus genogroups, but at both ends of the protein there are regions that stay largely the same. This suggests that these are probably key components of the protein, since the lack of variation indicates that any changes to those areas would prevent the virus from reproducing or infecting new cells. With this in mind, the authors focus on these two regions as they search for the function of VP2.

 

Connecting the proteins

            The authors looked for an interaction between the major capsid protein VP1 and VP2 at the N-terminal end of VP2 (the region shown on the left of Figure 2B). To start, they looked to see if connections between the proteins stayed if VP2 was shortened. The authors found that if VP2 was shortened by more than 50 amino acids, the interaction with VP1 was lost. This told them that VP2 connected with VP1 at a location between the end of the protein and 50 amino acids in. 

 

Figure 3B: The N-terminal end of VP2 is the location of interactions with VP1. In this immunoprecipitation experiment, VP1 and VP2 are added to a mixture together and allowed to interact before VP1 is removed from the mix with anything that is attached to it. This figure shows that VP2 is found attached to VP1 when the N-terminal end of the protein is intact (represented by the green bars in the second row down) but when everything before the 50^th amino acid is lost VP2 no longer interacts with VP1. This shows that the site where VP2 and VP1 interact is before the 50^th amino acid in VP2.

            After determining the general area where the VP1-VP2 interaction happens, the authors then focus on isolating the specific amino acids on VP2 that connect with VP1. They find that changing amino acids 40-43 causes the mutated VP2 to lose its interaction with VP1, suggesting that these amino acids are where the interaction occurs. Further investigation suggests that all amino acids in this region contribute to the interaction and all of them need to be mutated for the interaction between VP1 and VP2 to be completely lost. 

 

Figure 3C: Mutations to amino acids 40-43 in VP2 cause the protein to lose its interaction with VP1. The same experimental procedure is followed as in 3B. Again, VP1-Vp2 interaction is represented by the presence of green bars in the second row down. These bars disappear when amino acids 40-43 are changed, but not when amino acids 39-42 are.

            Next, the authors investigated how the interactions between VP1, VP2, and VPg that form the norovirus capsid occur. The authors first confirm that the same region responsible for VP1-VP2 interaction, amino acids 40-43 on VP2, is not also the place where VPg interacts with VP2. They also show that VP1 and VPg cannot interact on their own. In figure 5C, the authors localize the interaction between Vp2 and VPg to the C-terminal end of VP2. This suggests that VP2 might act as a bridge between the two other capsid proteins, with interactions at one end connecting VP1 and capturing VPg with the other end. 

 

Figure 5C: VP2 interacts with VPg at the C-terminal end. Using a similar experiment to figure 3B, the authors see if VPg (represented by the blue band in the second row down) can interact with either the first or the second half of VP2. They find that when only the first half of VP2 is present VPg cannot bind, but VPg can attach when only the second half of VP2 is present.

            The final question the authors look into is whether VP2 can interact with both VP1 and VPg at the same time. Using the same type of experiment as before, they find that when VP2 is intact and present, pulling VP1 out of a sample also pulls out VP2 and VPg despite the established lack of direct interaction between VP1 and VPg. This confirms the authors’ hypothesis that VP2 bridges the gap between the other two capsid proteins, allowing the norovirus capsid to form.

 

But how does this keep me from getting food poisoning?

            For all the discomfort norovirus causes around the world, we know surprisingly little about it. The more we know about this virus, the more chances we will have to develop an effective vaccine to prevent disease or treatments for after people get sick. Understanding the norovirus capsid is particularly important for this. Viral capsids are often what gets noticed by the immune system (and what we can engineer an immune response against for a vaccine). Additionally, without an intact viral capsid the virus cannot spread to a new cell. Early in the paper, the authors mention that without intact VPg norovirus is not infectious. If we can figure out a way to inactivate VPg with a medication, it could help us limit and treat severe norovirus cases. This work is an important step forward in understanding the function of the proteins in the norovirus capsid, and lays the groundwork for future discoveries towards vaccine or antiviral design. While there is much still to be learned, understanding the basics of how the norovirus capsid is built is a crucial development in the field.