Research

Research summary

Research in the Lindenbach laboratory focuses on the replication of hepatitis C virus (HCV) and related positive-strand RNA viruses. Specifically, we combine genetic, biochemical, and cell biological approaches to study how viral structural and nonstructural (NS) proteins contribute to RNA replication and virus particle assembly.

Virus assembly

We identified a number of conserved residues in the HCV NS2 protein that are important for virus particle assembly [1]. Specifically, mutations at these sites yielded viral genomes that could replicate in RNA-transfected cells but were unable to produce infectious virus particles. We then selected for revertant viruses that overcame these defects, which revealed genetic interactions between NS2 and the E1-E2 glycoprotein and NS3-4A enzyme complexes that are important for virus assembly. To follow up, we developed methods to biochemically capture NS2-containing complexes from virus-producing cells, which confirmed that NS2 forms critical interactions with the E1-E2 and NS3-4A complexes [2]. More recently, we have undertaken a similar approach to examine the role of NS3-4A in virus particle assembly and to map residues that mediate interaction with NS2.

Model of HCV assembly, adapted from [3].
Model of HCV assembly, adapted from [3].

To examine the cell biology of HCV particle assembly in greater detail, we developed methods to fluorescently label functional core protein in virus-producing cells [3]. These data showed that core protein is rapidly trafficked to the surface of lipid droplets, which appear to associate with sites of virus assembly near the interface between the endoplasmic reticulum (ER) and lipid storage droplets. After egress from lipid droplets, core protein is incorporated into virus particles that traffic through the secretory pathway. By examining core trafficking in our NS2 mutants, we showed that the interaction between NS2 and NS3-4A is essential for recruiting core from the surface of lipid droplets into virus assembly sites. Our current working model is that the interaction between NS2 and NS3-4A regulates the flow of RNA out of replication and into packaging (see Figure).

RNA replication

For many HCV NS proteins, biochemical activities have been characterized and several high-resolution crystal structures are available. However what we most lack is an understanding of how these pieces work together to form the active replication complex, and how host cofactors influence the steps of translation and replication. In collaboration with Dr. Anna Pyle, we are combining genetic and biochemical approaches to close this gap in our knowledge. Specifically, we are defining critical interactions between the serine protease and RNA helicase domains of NS3-4A [4], and examining how changes in NS3-4A conformation correlate with function. Furthermore, we have developed a novel trans-complementation system to dissect the assembly of functional replication complexes.

Long-term goals, the big picture

We have established a track record of accomplishment for our work on HCV particle assembly, and are making great progress on HCV RNA replication and innate antiviral defense. One long-term goal is to extend our studies to other members of the Flaviviridae to identify common themes and differences within this virus family. To this end, we have initiated studies to understand the role of NS proteins of yellow fever virus and dengue virus in replication and virus assembly. A second long-term goal is to develop new reagents to study HCV. To this end, we are developing novel methods to rapidly screen and identify additional HCV isolates that replicate in cell culture.

 

References

  1. Phan, T., et al., Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol, 2009. 83(17): p. 8379-95.
  2. Stapleford, K.A. and B.D. Lindenbach, Hepatitis C Virus NS2 Coordinates Virus Particle Assembly through Physical Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes. J Virol, 2011. 85(4): p. 1706-17.
  3. Counihan, N.A., S.M. Rawlinson, and B.D. Lindenbach, Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly. PLoS Pathog, 2011. 7(10): p. e1002302.
  4. Beran, R.K., B.D. Lindenbach, and A.M. Pyle, The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase. J Virol, 2009. 83(7): p. 3268-3275.
  5. Luo, D., et al., Structural Insights into RNA Recognition by RIG-I. Cell, 2011. 147(2): p. 409-22.