The long-term goals of the research efforts within our laboratory have been to define the biochemical and immunological properties of selected herpes simplex virus (HSV) glycoproteins and tegument proteins and to provide new insight regarding their synthesis, processing, interactions, and functional roles within HSV-infected cells. As a significant extension to these studies, the overall objective of this competitive renewal application is to define the roles HSV-1 tegument and integral membrane proteins play in the envelopment/budding process. The working hypothesis for the proposed studies is that one or more specific tegument proteins are major participants in the initiation of the envelopment process (i.e. budding). It is also part of the working hypothesis that enveloped viruses in general are likely to use similar mechanisms to accomplish the envelopment process. The expertise and experience of two laboratories (Courtney and Wills) will join to address the goals of the project. The Courtney laboratory has for a number of years studied various aspects of HSV glycoproteins and tegument proteins. The Wills laboratory has defined, at the molecular level, specific amino acid domains that are responsible for the envelopment and budding of retroviruses. The proposed studies includes four specific aims that are interrelated, but are independent of each other.
The first aim will focus on the expression and targeting of individual tegument proteins. Our working hypothesis is that targeting information within these tegument proteins plays a key role in the tegumentation of virus particles at internal membranes within virus-infected cells. These studies will dissect the targeting domains of tegument proteins and provide new insight as to what controls their localization within the cell.
The second aim will-focus on the identification of the minimal budding machinery of HSV-1. The working hypothesis is that a subset of tegument/membrane proteins has the potential to mediate the envelopment/budding process. Two experimental approaches will be used. The first is to express selected tegument proteins, both individually and m various combinations and to determine if these proteins can mediate envelopment and egress of virus particle/vesicles into the media. In the second approach, the Rous sarcoma virus Src membrane-binding domain fused to tegument proteins will be used to target these proteins to the plasma membrane. This approach will allow us to investigate envelopment and release of enveloped particles from the plasma membrane and thus bypass events associated with the egress of enveloped particles to the extracellular space.
The third aim focuses on the molecular dissection of two tegument proteins as an approach to define the packaging events that occur during assembly.
The fourth aim will focus on the identification of retroviral M (membrane binding) and L (late) domain equivalents within HSV-1 tegument proteins. Recent studies have suggested that late domains interact with specific domains of cellular proteins and are required for the virus-cell separation event (""""""""pinching off').
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