We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this

Glycoprotein gD is a component of the herpes simplex virus (HSV) envelope essential for virus entry into susceptible cells. Previous studies using deletion and point mutations identified a functional domain of HSV-1 gD (gD-1) from residues 231 to 244. However, many of the deletion mutations had global effects on gD-1 structure, thus precluding assessment of the functional role of large portions of the protein. In this study, we constructed a large panel of linker-insertion mutants in the genes for gD-1 and HSV-2 gD (gD-2). The object was to create mutations which would have only localized effects on protein structure but might have profound effects on gD function. The mutant proteins were expressed in transiently transfected L cells. Monoclonal antibodies (MAbs) were used as probes of gD structure. We also examined protein aggregation and appearance of the mutant glycoproteins on the transfected cell surface. A complementation assay measured the ability of the mutant proteins to rescue the infectivity of the gD-null virus, FgDI, in trans. Most of the mutants were recognized by one or more MAbs to discontinuous epitopes, were transported to the transfected cell surface, and rescued FgDi virus infectivity. However, some mutants which retained structure were unable to complement FgDi8. These mutants were clustered in four regions of gD. Region III (amino acids 222 to 246) overlaps the region previously defined by gD-1 deletion mutants. The others, from 27 through 43 (region I),

Four partially overlapping mRNAs (1.9, 2.3, 3.9, and 4.5 kilobases [kb]) were located between 0.16 and 0.19 map units on the herpes simplex virus type 1 genome. Their direction of transcription was found to be from right to left. The 2.3-kb mRNA was found to be early (1), whereas the others were late (P3y). Partial sequence analysis of the DNA encoding these genes indicated that the promoter for the 2.3-kb mRNA shares structural features with other early (1) promoters. In vitro translation of hybrid-selected mRNA indicated that among the proteins these mRNAs encode are an 82,000-dalton (d) polypeptide reactive with a monoclonal antibody against herpes simplex virus type 2 alkaline exonuclease and a 50,000-d polypeptide weakly reactive with a polyclonal antibody made against the capsid protein VP19C. Further experiments suggested that the 2.3-kb mRNA encodes the 82,000-d polypeptide, whereas one (or both) of the larger mRNAs encodes the 50,000-d protein. A novel finding was that the 1.9-kb mRNA appears to share part of the translational reading frame for alkaline exonuclease, but any polypeptide it encodes does not react with the monoclonal antibody to this enzyme.

All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.

The two partially colinear 6-kilobase (kb) and 1.5-kb mRNAs mapping between 0.23 and 0.27 map units on the herpes simplex virus type 1 genome were precisely located. The 5' end of the 6-kb mRNA was located 28 bases downstream of the sequence ATATATT and was 10 bases to the left of the BamHI site at 0.268. This position is ca. 90 bases to the left of our earlier reported sequence (R. J. Frink, K. G. Draper, and E. K. Wagner, Proc. Natl. Acad. Sci. U.S.A. 78:6139-6143, 1981). We used a polyclonal antibody made against purified herpes simplex virus type 1 VP5 to demonstrate that the 155,000-dalton translation product of the 6-kb mRNA is this capsid protein. The antibody did not react with the 35,000-dalton translation product of the 1.5-kb mRNA. We also confirmed our identification of VP5 as the translation product of the 6-kb mRNA by comparison of tryptic peptides of the in vitro-translated protein and authentic VP5. Recent work from this and other laboratories (reviewed in reference 22) has shown that herpes simplex virus (HSV) genes are generally encoded by unspliced transcripts under individual promoter control. A high-resolution location for a viral transcript, then, closely approximates the location of a specific gene. We previously mapped an extremely abundant 6-kilobase (kb) HSV type 1 (HSV-1) mRNA between 0.23 and 0.27 map units on the viral genome (6). We suggested that this is the mRNA for the major HSV-1 capsid protein (VP5) because it encodes a 155,000-dalton (Da) polypeptide in translation experiments in vitro and because it maps near the location for this gene determined from intertypic recombinant studies (17, 19). A second mRNA of 1.5 kb is colinear with the 3' portion of the 6-kb mRNA and translates a 35,000-Da polypeptide in vitro. The relationship between the two proteins is unknown. Our best available mapping data are summarized in Fig. 1A. This communication shows that we have carefully determined the position of the 3' and 5' ends of these two colinear mRNAs. In the case of the 6-kb mRNA, this positioning revealed that our earlier location for the 5' end (11) is in error and actually reflected the 5' end of a very minor transcript of unknown function. Our data demonstrate that the 5' end of a 3.1-kb mRNA transcribed off the opposite strand is very close to the 5' end of the 6-kb mRNA. We used a polyclonal antibody prepared against purified VP5 (NC-1; 5) to precipitate the 155,000-Da in vitro translation product of the 6-kb mRNA. This antiserum did not react with the 35,000-Da polypeptide. Finally, we fully confirmed the identity of the 155,000-Da in vitro translation product by comparing its tryptic peptides with those previously reported for purified VP5 (5, 20).