Research

VIRUS ENTRY AND EXIT

The research in our lab focuses on the molecular mechanisms of virus-membrane fusion and budding, using primarily the
alphavirus Semliki Forest virus (SFV) and the flavivirus dengue virus (DV).


W
hy are these viruses impo
rtant?

Flaviviruses and alphaviruses cause severe human and animal illnesses such as e
ncephalitis and hemorrhagic fever. Many of these viruses are pathogens identified by the Centers for Disease Control as being especially important agents of emerging infectious diseases and/or potential bioterrorist agents (category A-C pathogens). These include the flaviviruses dengue, West Nile, Japanese encephalitis and yellow fever viruses, and the alphaviruses Venezuelan, eastern, and western equine encephalitis viruses. Dengue virus (DV), a category A pathogen, is of particular concern as it has dramatically reemerged to become endemic in >100 countries, with an estimated 100 million cases of dengue infection per year. 

Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. SFV has been a critical model system to study the alphaviruses and flaviviruses since it is highly defined biochemically and structurally, has a very robust infection cycle and an easily manipulated infectious clone, and has low pathogenicity.



How do alphaviruses and flaviviruses enter cells?


The entry of both alphaviruses and flaviviruses takes advantage of cellular endocytosis, the pathway used by cells to take up many extracellular molecules such as hormones and nutrients. Virus binds to receptors on the plasma membrane and is then
internalized by endocytosis and delivered to the endosome compartment.

Endosomes have a mildly acidic pH that acts to trigger the fusion of the virus membrane with that of the endosome.





We know a lot about what happens during low pH-triggering.



















(a) The alphavirus membrane contains a dimer of 2 transmembrane glycoproteins, E2 (shown in cyan) and E1 (shown in color).

(b) Low pH causes the dissociation of the E2/E1 heterodimer, releasing E1 from its regulation by E2.

(c) E1 is the membrane fusion protein and inserts into the target membrane in a cholesterol-dependent reaction.

(d, e, f) E1 then rearranges to form a highly stable E1 homotrimer that has a “hairpin”, folded-back conformation. The conformational transition to the E1 trimeric hairpin brings the virus and target membranes together and drives membrane fusion.

In collaboration with Dr. Félix Rey and his colleagues, we determined the structure of the homotrimer conformation of SFV E1. Based on this structural and functional information, we can now address how the membrane fusion process works at the molecular level.



Inhibition of alphavirus and flavivirus fusion.

The flavivirus and alphavirus membrane fusion proteins are structurally and functionally similar and are therefore grouped together as members of the class II virus fusion proteins. Using the structures as a guide, our lab has recently developed fragments of the SFV and DV fusion proteins that act as dominant-negative inhibitors of SFV and DV fusion and infection.


Based on this information, we are working to establish general screens for inhibitors of class II fusion reactions. Such inhibitors will serve as lead compounds to develop new antiviral therapies.





How does virus budding work?


Our understanding of the pathways by which enveloped viruses assemble and bud is very limited, even though these are key steps in the production of new infectious virus particles. Virus budding can be targeted to the membranes of various cellular compartments including the endoplasmic reticulum, Golgi, nucleus and plasma membrane. Budding of different viruses also differs in requirements for membrane proteins and/or the virus core.



Budding of the alphaviruses (see lifecycle picture) occurs at the plasma membrane, is dependent on the specific interaction of the E2 cytoplasmic tail with the viral nucleocapsid, and produces highly organized virus particles containing 240 copies each of E2, E1 and capsid. How does this happen and what controls budding? Is alphavirus budding a self-driven process or is it dependent on cellular energy, chaperones, and other components? We have developed assays for the budding of cell surface E2/E1 into alphavirus particles, enabling us to address the mechanism of budding.


What are the important questions to address next?


This is an exciting time for studies of virus entry and exit. There are many important areas of research, including the molecular mechanism of the homotrimer during membrane fusion, the identification and use of fusion inhibitors, mutagenesis of virus infectious clones to characterize fusion protein function, the mechanism of E1-membrane insertion and the role of cholesterol, the structure and function of the E1 transmembrane and stem regions, the regulation of virus budding and the role of cellular factors in budding.