Bridging KSHV capsids to the nuclear egress complex

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DESCRIPTION (provided by applicant): The herpesvirus virion is comprised of three structural components: an icosahedral capsid, which encloses the genome; an electron dense asymmetrically distributed material, which immediately surrounds the capsid and is termed the tegument; and an outer membrane or envelope, which encloses the tegument and capsid and in which are embedded the viral glycoproteins. These virions are the most complex, highly organized structural entities comprised of several protein sub-units. Two studies on Kaposi's sarcoma-associated herpesvirus (KSHV) from our lab have accomplished the self-assembly of icosahedral capsids and re-constitution of the nuclear egress complex (NEC), in insect cells using baculoviruses for protein expression. This has given us a unique opportunity and the impetus to do something novel and innovative that could have the potential to be translated into the development of virus-like particles (VLPs) for new vaccine platforms. The aims will focus on beginning to physically link KSHV capsids with the NEC using biochemical and cell biological methods in this "ex-vivo" culture system. We will test capsid-associated proteins, for example, the capsid vertex-specific component (CVSC) encoded by open reading frames (ORF) 19 and 32 for the ability to bridge capsids with nuclear membranes. The outcomes will also shed light on the mechanism of capsid maturation for KSHV and for other herpesviruses. Specific Aim 1. Elucidate the interactions and capsid association of the KSHV CVSC. (a). Interactions of ORF32 and ORF19 with the capsid proteins. (b). Binding of the CVSC to KSHV icosahedral capsids. Specific Aim 2. Discover how to physically link KSHV capsids with the NEC. (a). Interactions of the NEC with the CVSC. (b). Link assembled capsids with the NEC in nuclear membranes. This proposal will discover if the "ex-vivo" produced KSHV capsids can be "decorated" by the CVSC and then test these for their potential to bind to the nuclear egress complex. The final goal of this proposal is to link these structural entities together. The outcome of this approach if successful is the use of these particles for new vaccine platforms for a clinically important pathogen and to develop new antiviral strategies.