Identification of a domain of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation through the use of protein A fusion proteins.

1989 ◽  
Vol 63 (12) ◽  
pp. 5509-5513 ◽  
Author(s):  
G Werstuck ◽  
J P Capone
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Complex Formation ◽  
Fusion Proteins ◽  
Protein A ◽  
Simplex Virus ◽  
Dna Complex

scholarly journals Origin Binding Protein-Containing Protein-DNA Complex Formation at Herpes Simplex Virus Type 1 oriS: Role in oriS-Dependent DNA Replication

2001 ◽  
Vol 75 (15) ◽  
pp. 6808-6816 ◽  
Author(s):  
Jennifer A. Isler ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Complex Formation ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Origin Binding Protein ◽  
Simplex Virus ◽  
Dna Complex

ABSTRACT Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.

scholarly journals Mutational Evidence of Internal Fusion Loops in Herpes Simplex Virus Glycoprotein B

2007 ◽  
Vol 81 (9) ◽  
pp. 4858-4865 ◽  
Author(s):  
Brian P. Hannah ◽  
Ekaterina E. Heldwein ◽  
Florent C. Bender ◽  
Gary H. Cohen ◽  
Roselyn J. Eisenberg
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Proteins ◽  
Glycoprotein B ◽  
Functional Domain ◽  
Virus Glycoprotein ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is one of four glycoproteins necessary and sufficient for HSV cellular entry. Recently, the crystal structures of HSV-1 gB and vesicular stomatitis virus glycoprotein G were determined. Surprisingly, the two proteins share remarkable structural homology. Both proteins are homotrimeric and center about a long alpha-helix, features reminiscent of class I fusion proteins, such as influenza virus hemagglutinin or paramyxovirus F. However, these structures revealed that G has internal fusion loops, similar to the fusion loops of the class II fusion proteins, and that these loops are structurally conserved in gB. To examine whether these putative fusion loops are important for gB function, we mutated potential membrane-interacting (hydrophobic) residues to charged amino acids. Of most interest were mutant gB proteins that were expressed on the cell surface and were recognized by monoclonal antibodies against conformational epitopes but lacked the ability to function in cell-cell fusion assays. We find that three of the five hydrophobic amino acids targeted in these loops, tryptophan 174, tyrosine 179, and alanine 261, are integral in the function of gB. Our data suggest that they are part of an important functional domain. We hypothesize that two loops in domain 1 of HSV gB function as fusion loops. Our data are further evidence that gB is a viral fusogen and suggest clues as to how gB may function.

scholarly journals Reevaluating Herpes Simplex Virus Hemifusion

2010 ◽  
Vol 84 (22) ◽  
pp. 11814-11821 ◽  
Author(s):  
Julia O. Jackson ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Viral Fusion Proteins ◽  
Simplex Virus

ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

scholarly journals Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

Virology ◽  
2014 ◽  
Vol 460-461 ◽  
pp. 128-137 ◽  
Author(s):  
Martina Maric ◽  
Alison C. Haugo ◽  
William Dauer ◽  
David Johnson ◽  
Richard J. Roller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Herpes Simplex Virus Type ◽  
Fusion Proteins ◽  
Viral Fusion ◽  
Nuclear Envelope Breakdown ◽  
Viral Fusion Proteins ◽  
Simplex Virus
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