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Tsvitov, Marianna (2006) CHARACTERIZATION OF SOLUBLE HERPES SIMPLEX VIRUS TYPE 1 GLYCOPROTEIN D MEDIATED INFECTION. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The predominant mechanism of herpes simplex virus type 1 (HSV-1) entry into permissive cells involves initial virus attachment to the cells by the interaction of envelope glycoproteins gC and gB with cell surface glycosaminoglycans (GAGs), binding of envelope glycoprotein D to one of several dissimilar co-receptors, and fusion of the virus envelope with the cell membrane requiring the combined essential functions of glycoproteins gD, gB and gH/gL. The binding of gD to its cognate receptor appears to result in emission of an activating signal to the fusion apparatus which minimally consists of the other essential glycoproteins. To gain a better understanding of gD's involvement in the fusion-activating process, we took the approach of separating gD from the virus envelope to determine whether a soluble form of gD (sgD) could mediate entry of gD-deficient virus. The results showed that sgD enabled entry of gD-deficient HSV-1 into CHO-K1 cells bearing the gD receptors HVEM or nectin-1. Using mutant forms of sgD that selectively bind to one or the other receptor, we demonstrated that entry by this mechanism is receptor specific. Investigation of the mechanism of sgD-mediated entry demonstrated that the presence of virus at the cell surface was required at the time of sgD-receptor binding, which could be explained in part by our observation that sgD rapidly dissociated from the receptor under our experimental conditions. In addition, entry was not eliminated instantaneously when receptor-bound sgD was exposed to 37ºC, suggesting that the active conformation of receptor-bound sgD is not highly unstable. sgD was not stabilized at the cell surface or internalized in the presence of gD-deficient virus. Using lysosomotropic agents as well as protease protection assays, we obtained no reproducible evidence that sgD-mediated entry takes place by endocytosis.Surprisingly, virus attachment to cell-surface GAGs was not required for sgD-mediated entry. Furthermore, gD-deficient virus attached to GAG-deficient cells in the absence of sgD, revealing a previously unknown binding interaction between the HSV virion and the cell. This interaction was shown to be of a less stable nature than the virus-GAG interaction, and may play a role in normal virus entry. Our results provide new tools and directions to unravel the still incompletely understood events set in motion by gD binding to its receptor.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Tsvitov, Mariannamat35@pitt.eduMAT35
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGlorioso, Joseph Cglorioso@pitt.eduGLORIOSO
Committee MemberHoma, Fred Lflhoma@pitt.eduFLHOMA
Committee MemberWeisz, Ora Aweisz@pitt.eduWEISZ
Committee MemberRobbins, Paul Dprobb@pitt.eduPROBB
Committee MemberKinchington, Paul
Committee MemberPhillips, Stephen Lsleep@pitt.eduSLEEP
Date: 17 August 2006
Date Type: Completion
Defense Date: 29 September 2005
Approval Date: 17 August 2006
Submission Date: 3 August 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: gD; glycoprotein D; Herpes Simplex Virus Type 1; HSV; HSV entry; soluble gD
Other ID:, etd-08032006-110604
Date Deposited: 10 Nov 2011 19:56
Last Modified: 15 Nov 2016 13:47


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