Brett Lindenbach, PhD

Many RNA viruses encode RNA helicases that are essential for viral replication, and it is frequently assumed that these enzymes unwind double-stranded forms of the viral gemone. Indeed, many of these enzymes have been shown to have RNA binding, ATPase, and RNA unwinding activities in vitro. However, to date there is no direct evidence that these enzymes bind to or unwind viral RNA in infected cells. We recently identified several important activities of the HCV NS3-4A RNA helicase domain in recruiting RNA an template for replication and in in virus assembly.

RNA replication

For many HCV nonstructural (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. We are combining genetic and biochemical approaches to close this gap in our knowledge. Specifically, we have developed a novel trans-complementation system to dissect the features of viral NS proteins proteins required for assembly of functional replication complexes. By using this system, we discovered the HCV NS3-4A helicase recruits the viral genome in cis (i.e., the same RNA from which it is translated) out of translation and into RNA replication. We also found that NS5B has an essential cis-acting role in RNA replication, independent of its RNA binding and RNA polymerase activities. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.

Virus assembly

We discovered that the HCV NS2 protein interacts with both the viral E1-E2 glycoprotein complex and the NS3-4A enzyme complex and that these interactions are essential for virus particle assembly. 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. Our data revealed that core protein is rapidly trafficked to the surface of lipid droplets, which associate with the sites of virus assembly at the ER. After egress from lipid droplets, core protein is incorporated into virus particles, which bud into the ER and traffic via the secretory pathway. By examining core trafficking in NS2 mutants with or without second-site genetic suppressors in NS3, we showed that the interaction between NS2 and NS3-4A is essential for recruiting core from the surface of lipid droplets into virus particles. Our current working model is that the interaction between NS2 and NS3-4A regulates the flow of RNA out of replication and into packaging. Because RNA helicase activity is essential for RNA replication, which is a pre-requisite for virus assembly, we have developed a unqiue genetic approach to separate the functions of the NS3 helicase in viral genome replication from its role in virus assembly.

Bacterial effectors as probes to study (+) RNA virus-host cell biology

The mechanisms by which viruses interact with their host cells are incompletely understood; identifying these interactions remains a fundamentally important area of basic virus research. The three most common approaches to discovering virus-host cell interaction have been: 1) Genome-wide RNAi screens for host genes that influence viral replication; 2) Identifying protein-protein interactions via proteomics or genetic two-hybrid screens; 3) Screening pharmacological agents to disrupt known cellular pathways. While these approaches have been incredibly useful, their limitations include: variability in RNAi knockdown efficiency, off-target effects, limited reproducibility between genome-wide screens, false-positive scoring of protein-protein interactions, and a relatively small and nonspecific pharmacopeia. We are exploring a new strategy to identify virus-host cell interaction by employing a large collection of bacterial effector proteins as a genetic toolkit to surgically manipulate key cellular pathways. Many bacterial pathogens infect and survive within eukaryotic cells by injecting minute quantities of bacterial effector proteins, typically enzymes, into the cytosol of their hosts. These effector proteins have evolved to manipulate cellular pathways, prevent bacterial degradation, and favor bacterial replication. For instance, Legionella pneumophila, the causative agent of Legionnaire’s disease, synthesizes >300 effector proteins, some of which reprogram endolysosomal membrane trafficking, potently inhibit cellular autophagy, and divert innate immune responses. Importantly, many effector proteins retain their function when ectopically expressed in mammalian cells and can be used to study cellular pathways independent of bacterial infection. Bacterial effector proteins frequently target the same cellular pathways used by (+) RNA viruses. For instance, Legionella effector proteins manipulate Rab1, a key organizer of ER-to- Golgi membrane traffic and a host factor required for hepatitis C virus (HCV) replication. Other effectors inhibit autophagy, a pathway exploited by HCV and many other (+) RNA viruses. Based on these known functional overlaps we hypothesize that bacterial effector proteins can be used as tools to identify cellular pathways used by (+) RNA viruses.