Associate Professor, Molecular Biosciences
The University of Kansas
Mechanisms of Genome Packaging in DNA Viruses (2010-12)
Human herpesviruses are important pathogens that cause diseases ranging from mucocutaneous lesions, retinitis to encephalitis, and various forms of cancer. The terminase and portal in herpesviruses have been used as drug targets. The proposed studies can contribute to public health by opening avenues towards novel measures to prevent or cure diseases caused by herpesviruses.
Genome encapsidation is a crucial process in virus lifecycles, and is essential for assembly of infectious progeny virions. Various mechanisms are employed by DNA and RNA viruses to achieve this. In herpesviruses and many tailed double-stranded DNA (dsDNA) bacteriophages, this process is fulfilled in a precisely coordinated molecular synergy involving a powerful molecular device that pumps viral DNA into a preformed protein shell called procapsid. The viral DNA packaging device consists of a portal protein and a two-component enzyme complex called terminase. The portal forms a conduit at a single vertex of the capsid that allows viral DNA to enter for virus assembly and exit for infection. The terminase contains a DNA-recognition subunit (also known as small subunit) that specifically binds to the viral DNA, and a catalytic subunit (a.k.a. large subunit) that provides the energy for the packaging reaction via ATP hydrolysis and cleaves genome-length units from the viral DNA concatemer. In host cells, the terminase recognizes viral DNA concatemer, docks onto the portal, and inserts a certain amount of viral DNA into the procapsid, followed by a series of later events such as cleavage of DNA, detachment of the terminase, and binding of additional viral proteins to retain the packaged DNA.
DNA packaging proteins form high-order molecular assemblies, and molecular assembling occurs in a carefully controlled, hierarchical manner, which is essential for successful viral DNA packaging. No high resolution structural information is available for herpesvirus DNA packaging proteins. Little is known about assembly among terminase subunits, the portal and the capsid. Our long-term goal is to understand molecular mechanisms of DNA packaging in dsDNA viruses at high resolution. In this proposal, we seek to perform biochemical and crystallographic studies on (i) the HSV-1 terminase catalytic subunit pUL15 and its homolog pORF29 in Kaposi’s sarcoma associated herpesvirus, and (ii) the complete Sf6 virion. These studies are anticipated to lead to high resolution structural analysis.
Signal Transduction of Two-Component System (2008-2010)
For bacteria, the ability to sense and adapt to environmental change and stress is crucial to persistence and survival. Two-component system (TCS) is a fundamental mechanism utilized by bacteria as well as fungi and some plants to sense external or internal signals and make appropriate responses through regulation of gene expression. TCSs play pivotal roles in pathogenesis and/or antibiotic resistance in many pathogenic microorganisms. A paradigmatic TCS consists of two proteins: a multi-domain, multi-function sensor histidine kinase (HK) that monitors environmental signals, and a response regulator (RR) that receives the signal from the sensor kinase through phosphotransfer reactions and trigger downstream responses through, e.g., regulation of expression of certain genes. Molecular mechanisms underlying TCS signal transduction remain poorly understood.
The proposed research is aimed at molecular basis of TCS signal transduction at high resolution, using the YycFG of Bacillus subtilis as a model. YycFG is an essential TCS and is specific to low G+C Gram-positive bacteria such as Staphylococcus, Streptococcus and Enterococcus, which are leading causative agents of human infections and cause diseases ranging from pneumonia to meningitis, endocarditis and bacteremia. Some of these pathogens, such as Bacillus anthracis and Clostridium botulinum, are classified as potential bioterror agents. We seek to:
- explore the domain organization of YycG at high resolution by determination of the structure of a truncated form of YycG,
- define the three-dimensional architecture of YycF, and
- investigate the molecular interactions between YycG and YycF that are critical for phosphotransfer.