A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1.

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1.

A glutathione S-transferase fusion to the COOH-terminal acidic transactivation domain of Vmw65 from herpes simplex virus type 1 was overexpressed in Escherichia coli and isolated by affinity chromatography on glutathione-Sepharose. Following cleavage of the fusion protein with thrombin, the transactivation domain was purified to homogeneity by ion exchange chromatography yielding approximately 0.6 mg of protein/liter of bacterial culture. Equilibrium sedimentation analysis showed the purified polypeptide to be monomeric; however, it displayed aberrant electrophoretic and chromatographic properties. Contrary to secondary structure predictions, circular dichroism spectroscopy demonstrated that this transactivation domain was devoid of significant alpha-helical structure at physiological conditions. The polypeptide, however, became notably more structured under hydrophobic conditions or at low pH, suggesting that it was sensitive to its environment. Near-UV circular dichroism suggested that phenylalanyl and tyrosyl residues were under influence from tertiary structure.     

Mutational analysis of the herpes simplex virus triplex protein VP19C

Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal approximately 100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.     

The herpes simplex virus protein Vmw65 can trans-activate both viral and cellular promoters in neuronal cells.

Somatostatin inhibited Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells, albeit with decreased sensitivity relative to intact cells. The inhibitory action required the presence of GTP, whereas GDP could not substitute for GTP. Pertussis-toxin treatment before cell permeabilization abolished the inhibition of secretion. Thus somatostatin, by activating a G-protein, interferes with exocytosis distal to the generation of soluble intracellular messengers.     

Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR.

We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.     

Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65.

A transient expression system was developed which results in efficient synthesis of the regulatory protein Vmw65 of herpes simplex virus type 1 in eucaryotic cells. The gene for Vmw65 was linked to the cytomegalovirus immediate-early (IE) promoter-enhancer region in a plasmid containing the simian virus 40 origin of replication. When transfected into COS cells, Vmw65 was expressed from this vector in 25 to 50% of the cells, with total levels of the protein approaching 20% of those observed in infected cells. Vmw65 expressed in this system is functional for specific DNA-binding complex formation with the host cell octamer-binding protein TRF and for transactivation of IE gene expression. We therefore produced a series of carboxy-terminal truncated forms of Vmw65 to examine the structural requirements of the protein for these activities. Deletion of the acidic carboxy-terminal 56 amino acids had no effect on DNA-binding complex formation but completely abolished the ability to transactivate. Amino acids between residues 434 and 453, a region which exhibits a high negative charge, were critical for IE transactivation. In contrast, the requirements for complex formation are located entirely within the N-terminal 403 amino acids, and our results indicate a requirement for this activity for residues between 316 and 403. Together with our previous work, the results presented here indicate that recruitment of TRF into a specific DNA-binding complex on IE consensus signals is required but not sufficient for functional IE transactivation by Vmw65.     

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.

In order to identify structural domains of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation, subfragments of Vmw65 were expressed in Escherichia coli as fusion polypeptides with protein A of Staphylococcus aureus, and the purified hybrids were used in a band shift assay. The results indicate that a region near the amino terminus of Vmw65 between amino acids 141 and 185 is necessary for complex formation.     

Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B

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.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.