Department of Molecular and Cellular Biochemistry

Research in the Hardy laboratory is focused on the molecular requirements for virus replication. We are specifically interested in what molecules are necessary for the replication of the viral genetic material and the optimal expression of viral genes within host cells. Viruses present a unique challenge to the understanding of their replication because of their obligate, intracellular, parasitic nature; that is they can only replicate inside a host cell and require numerous cellular resources in order to proliferate. This intimate, though parasitic, relationship with the host cell means that we must look to both viral and host components to understand virus replication.

Cis-acting signals for virus replication and gene expression. Alphavirus genomic RNA replication is a two-step process; initially the plus-sense genomic RNA is copied into a full-length minus-strand intermediate, this minus-strand RNA then functions as a template for the production of multiple copies of the genome RNA. The replication enzymes required for these two processes are different while being derived from the same source. Upon infection the genome RNA functions as a messenger RNA and is translated to produce a polyprotein. A partially processed form of this polyprotein functions as the replicase for minus-strand RNA synthesis while a more completely processed form functions as the replicase for plus-strand synthesis. One of the major tools used in my laboratory is an in vitro minus-strand synthesis system. This system allows us to separate minus-strand RNA synthesis from translation or plus-strand synthesis, thus facilitating the analysis of mutational effects on this process alone. Using this method we have been able to identify cis-acting elements at both the 5’ and 3’ end of the genome that influence RNA replication. Additionally detailed molecular work has defined binding sites for the viral RNA polymerase in the 3’ region of the genome, and defined promoter functions within the genome. Future work will focus on how these cis-acting elements communicate with one another to coordinate the various functions carried out by the genome.

Replicase composition and assembly. An active are of research in the lab focuses on determining the molecular interactions required to form a functional genome replication complex. Which viral proteins contact each other? What host factors are part of this complex? Do host factors contact specific cis-acting elements? Using a sub cellular fractionation, proteomics, and immunoprecipitation techniques we have been able to identify multiple host proteins enriched in fractions containing active viral replication complexes. Initial analyses indicate some of these proteins are important for efficient virus replication. Additionally we have been able to establish specific interactions between viral components of the replication complex and continued work will aim to verify the significance of these interactions.

Virus-host interactions. Another research area in the lab is the examination of the virus-arthropod host interaction. Alphaviruses are obligatorily transmitted by a hematophagous arthropod vector in which a lifelong persistent infection is established. This pattern of infection implies an effective but incomplete immune response that preserves host fitness while allowing virus replication to continue thus facilitating virus transmission. A major impediment to understanding the critical virus-host interactions has been the inability to genetically manipulate the host organism. Drosophila melanogaster represents an excellent experimental system for elucidating the genetic, molecular, and biochemical mechanisms underlying numerous physiological and developmental processes. Our research utilizes the power of Drosophila genetics to define the recently characterized interactions between alphaviruses and two arthropod innate immune response pathways. We have established a system for the analysis of alphavirus replication in which an alphavirus replicon sequence encoding a reporter gene is expressed from the genome of Drosophila under the control of the GAL4-UAS misexpression system. The resulting replication of the viral RNA is observable through reporter gene activity. Mutational analyses of host pathways using this alphavirus replicon fly line have demonstrated an antiviral role for the Imd- and JAK-STAT pathways. This system has been further developed to produce infectious particles. Co-expression in Drosophila of the replicon and viral structural proteins leads to the production of replicon containing particles capable of a single round of infection. These basic tools are being used in combination with Drosophila genetics to (i) determine the viral components responsible for activation of Imd- and JAK-STAT pathways; (ii) identify host genes involved in virus-induced innate immune response; (iii) characterize host requirements for virus spread. Our research combines the power of Drosophila genetics with comparative genomics and molecular virology in order to advance our understanding of the interaction between alphaviruses and an arthropod host.