New insights into the mechanisms of lipid-body biogenesis in plants and other organisms.

A comparative approach has been used to study the role of several lipid-body-binding proteins in plants and animals. Caleosins are a newly discovered class of calcium-binding lipid-body proteins found in plants and fungi, which we now report to have separate endoplasmic reticulum- and lipid-body-associated isoforms. We also compare the lipid-body targeting of oleosin from plants and the core protein of the hepatitis C virus when they were expressed separately in lipid-accumulating animal cell lines. This is a novel and powerful approach to investigating the factors that determine the lipid-body targeting of a wide range of proteins.

Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system.

The DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17+ in the region coding for the polypeptide ICP34.5 predicts a protein of 248 amino acids with a proposed M(r) of 26,158. The entire RL1 open reading frame was cloned into the expression vector pET8c to enable over-expression of ICP34.5 in Escherichia coli. The expressed protein was partially purified and used as an immunogen to produce a polyclonal antiserum in rabbits. Construction of an ICP34.5 null mutant (1771), demonstrated that the predicted open reading frame for ICP34.5 in strain 17+ is correct and confirmed that HSV-1 strain 17+ ICP34.5 specifically determines neurovirulence. The specificity of the anti-serum directed against the E. coli-expressed ICP34.5 was defined by Western blotting of wild-type and RL1-negative infected cell extracts.

Specific transcriptional activation in vitro by the herpes simplex virus protein VP16.

The herpes simplex virus protein VP16 interacts with cellular factors, including the protein Oct-1, to activate viral immediate early (IE) gene transcription. We have reproduced this effect by addition of purified, full-length VP16 and the DNA-binding 'POU' domain of Oct-1 (Oct-1/POU) to a HeLa cell in vitro transcription system. Stimulation of transcription was dependent on the IE-specific element, TAATGARAT. In agreement with earlier observations from electrophoretic mobility shift assays, activation was not observed when Oct-2/POU, the DNA-binding domain from the Oct-2 protein, was substituted for Oct-1/POU. Single round transcription assays revealed that, together, VP16 and Oct-1/POU facilitate the assembly of pre-initiation complexes at target gene promoters.

Mapping of epitopes on the 65k DNA-binding protein of herpes simplex virus type 1.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

The 65,000-Mr DNA-binding and virion trans-inducing proteins of herpes simplex virus type 1.

The possible identity of the herpes simplex virus type 1 (HSV-1) 65K (65,000-Mr) virion protein which stimulates transcription from immediate-early genes with the HSV-1 65K DNA-binding protein was investigated. The two proteins were found to be distinct by the three separate criteria of immunological reactivity, tryptic peptide fingerprinting, and mobility in two-dimensional gels. Using HSV-1/HSV-2 intertypic recombinants and a serotype-specific antiserum, we located the gene encoding the 65K DNA-binding protein between coordinates 0.574 and 0.682 on the HSV-1 genome. The protein is posttranslationally modified by phosphorylation. In crude extracts of HSV-1-infected cells the 65K trans-inducing protein did not detectably bind to double-stranded calf thymus DNA under the conditions of our assay.

Generation and properties of the glycoprotein E-related 32K/34K/35K and 55K/57K polypeptides encoded by herpes simplex virus type 1.

A hybridoma line was isolated which produced antibody reacting with polypeptides of apparent molecular weights 32,000, 34,000 and 35,000 (32K/34K/35K) and 55,000 and 57,000 (55K/57K). These were sulphated glycoproteins that have previously been found in the medium of herpes simplex virus type 1 (HSV-1)-infected cells. By tryptic peptide mapping and serological cross-reactions the polypeptides were shown to be related to HSV-1 glycoprotein E (gE-1) but they lacked the Fc binding function. The 32K/34K/35K and 55K/57K polypeptides were not found in the medium of HSV-1-infected cells incubated in serum-free medium. They could be generated in vitro from purified gE-1 in the presence of serum. It is likely that 32K/34K/35K and 55K/57K are derived from gE-1 by the action of serum proteases.

Characterization of the 92,000-dalton glycoprotein induced by herpes simplex virus type 2.

Evidence is presented showing that the 92,000-dalton glycoprotein (g92K) induced by herpes simplex virus (HSV) type 2 has properties distinct from those assigned to any other HSV glycoprotein. First, the carbohydrate composition and extent of sulfation differ from those of glycoproteins D and E. Second, two clonally unrelated monoclonal antibodies, AP1 and LP5, shown in this paper to specifically immunoprecipitate g92K, do not react with any of the known processed forms of glycoproteins B, C, D, and E. Third, by using HSV type 1/HSV type 2 intertypic recombinants and a simple radioimmunoassay, the target antigen of the two monoclonal antibodies was shown to map in the same region as g92K (0.846 to 0.924). Fourth, the intertypic recombinant R12-3 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of infected cells to induce the HSV type 2 g92K and HSV type 1 gD and GE, whereas R12-1, which did not induce g92K, induced HSV-2 gE and an altered gD, providing genetic evidence that g92K is encoded, at least in part, by a different region of the genome from that encoding gD and gE.

Processing of glycoproteins induced by herpes simplex virus type 1: sulphation and nature of the oligosaccharide linkages.

Using the drug tunicamycin we have investigated the nature of the oligosaccharides on herpes simplex virus type 1 (HSV-1)-induced glycoproteins E and Y (gY is a newly identified glycoprotein which has the same apparent mol. wt. as gC but a more basic isoelectric point). Synthesis of both glycoproteins was inhibited by the drug, suggesting they contain N-linked oligosaccharides. Our finding, combined with the previous results of other workers, suggests that all the major HSV-induced glycoproteins have this type of carbohydrate modification. All of the major HSV-1-induced glycoproteins are modified by addition of inorganic sulphate. This modification occurs late in their maturation. Most inorganic sulphate appears to be attached to N-linked oligosaccharides but some is attached to other parts of glycoprotein E. Using HSV-1/HSV-2 intertypic recombinants, the mapping limits of that part of the glycoprotein E gene coding for differences in mobility between the two serotypes have been further narrowed and are located between coordinates 0.886 and 0.935.

Processing of herpes simplex virus type 1 glycoproteins: two-dimensional gel analysis using monoclonal antibodies.

The number of discrete species of glycoprotein induced by herpes simplex virus type 1 (HSV-1, strain 17 syn+) and their processing has been examined by a combination of immunoprecipitation with monoclonal antibodies and analysis of immune precipitates by two-dimensional (2D) gel electrophoresis. Seventeen different monoclonal antibodies directed against glycoproteins A/B, C, D and E were used. Polypeptides intermediate in the synthesis of gA/B, C and D were visualized and two early intermediates of glycoprotein A/B (pgA/B1 and pgA/B2), both mannose-containing, were identified. Comparison on 2D gels of polypeptides synthesized in vitro from HSV-1-infected cell mRNA with those synthesized in pulse-chase experiments in vivo has allowed identification of early precursors of pgA/B and pgD. Their apparent mol. wt. were 105000 and 47000 respectively. The 2D gel analysis of glycoproteins induced in HFL cells infected with 17 syn+ revealed a number of previously unreported glycoprotein species. One had mobility on both gradient and single concentration SDS-polyacrylamide gels similar to that of gC but was resolved from gC on non-equilibrium pH gradient gels. This glycoprotein was produced in relatively large amounts and was not precipitated by any of the monoclonal antibodies used in this study. The data suggest that this glycoprotein either has a polypeptide chain unrelated to those of glycoproteins A/B, C, D or E or alternatively is derived from one of them and modified in such a way as to mask or remove the antigenic sites with which the monoclonal antibodies interact. Sixteen other previously unreported glycoprotein species not obviously related, as judged by their electrophoretic mobility, to gA/B, C, D or E were also identified: two reacted with gA/B-specific monoclonal antibodies and five with glycoprotein C-specific monoclonal antibodies. The origin of the remaining nine new glycoproteins has still to be ascertained.

Genomic location and lack of phosphorylation of the HSV immediate-early polypeptide IE 12.

Herpes simplex virus type 1 (HSV-1) induces an immediate-early (IE) polypeptide IE 12. An equivalent polypeptide coded by HSV-2 which migrated slightly more slowly on SDS-polyacrylamide gels was identified and designated IE 12.3. Analysis of the serotype of the IE polypeptide induced by five HSV-1/HSV-2 intertypic recombinants when correlated with their genome structures showed that IE 12/IE 12.3 mapped in the region spanning the TRs/Us junction. Unlike four other IE polypeptides induced by HSV-1 (IEs 175, 110, 68 and 63), IE 12 was not detectably phosphorylated.

Sulphated glycoproteins induced by herpes simplex virus.

BHK cells infected with strain 17 syn+ (HSV-1) or HG52 (HSC-2) incorporated inorganic sulphate into polypeptides which co-migrated on SDS-polyacrylamide gels with virus-induced glycoproteins. The major sulphated glycoprotein was glycoprotein E. In addition, less-intense sulphated bands co-migrated with glycoprotein D and HSV-1 glycoprotein A/B/C. Sulphate label co-migrating with HSV-2 glycoprotein A/B/C was occasionally observed. We have investigated which sulphated polypeptides are excreted from infected cells. Major ones of apparent mol. wt. 32000, 34000 and 35000 were excreted from cells infected with syn+. In addition, polypeptides which migrated in the vicinity of glycoprotein D were often excreted from cells infected with either 17 syn+ or HG52. The 32K, 34K and 35K polypeptides were antigenically related to glycoprotein D and over 95% of the total amount synthesized was excreted. Analysis of intracellular sulphated polypeptides using intertypic recombinants mapped glycoprotein E to between 0.832 and 0.950 units of the HSV genome.