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We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Guinea pig antisera and mouse monoclonal antibodies against a 40,000-molecular-weight nucleocapsid protein (p40) of herpes simplex virus types 1 and 2 immunoprecipitated 40,000- and 80,000-molecular-weight classes of soluble proteins from infected cell extracts. The soluble 40,000-molecular-weight protein class (intracellular p40) appeared as a cluster of three to four closely spaced bands of proteins having molecular weights ranging between 39,000 and 45,000, whereas the soluble 80,000-molecular-weight protein class (intracellular p80) appeared as a doublet of bands. The peptide map of intracellular p40 closely resembled the maps of the p40 and p45 proteins of nucleocapsids, but it showed both differences and similarities when compared with the peptide map of intracellular p80. Pulse-chase experiments suggested that intracellular p80 was not a precursor of intracellular p40. We conclude that the intracellular p40 and p80 protein classes share common antigenic determinants, presumably reflecting similar amino acid sequences, although they have distinct differences in protein structure.

A polypeptide (p40) of approximately 40,000 molecular weight was isolated from herpes simplex virus type 1 and 2 nucleocapsids by gel filtration and ion exchange chromatography. This protein appears to be the same as protein 22a described previously (Gibson and Roizman, J. Virol. 10:1044--1052, 1972). Competition immunoassays were developed by using purified p40 and antisera prepared in guinea pigs. The assays indicated that the p40's from herpes simplex virus types 1 and 2 possess both type-specific and cross-reactive antigenic determinants. Antibodies to the p40 cross-reactive determinant reacted with antigens in simian herpes virus SA8-infected cells, but not with antigens induced by pseudorabies virus. Preliminary results indicated that a radioimmunoprecipitation test can be used to detect type-specific herpes simplex virus p40 antibodies in human sera.

Intact wild-type simian virus 40 particles can be separated and resolved from a temperature-sensitive mutant and from a number of other viruses by agarose gel electrophoresis. The relative mobilities of the viruses appear to be a function of both virion size and surface composition. The virions of a temperature-sensitive strain of simian virus 40, tsB204, have significantly greater mobility than those of wild-type simian virus 40, when electrophoresis was conducted toward the cathode at pH 5.0. When electrophoresis was performed toward the anode at pH 7.0, TSB204 viruses have a slightly slower mobility as compared with that of the wild type. The data indicated that the virions of tsB204 have a greater positive charge at their surface than those of wild-type particles. No differences were detected in the finger print patterns of the tryptic peptides of VP1 and VP3 of these two virus strains. Although it was not possible to identify the structural polypeptide directly affected by the tsB204 mutation, we have shown that this mutation affects charge distribution on the surface of the virion.

Herpes simplex virus type 1 (HSV-1) glycoprotein gC was purified by affinity chromatography with an immunosorbent column containing monoclonal antibody to HSV-1 gC, and its reactivity with rabbit antisera was measured by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of radioimmunoprecipitates. Positive reactions were detected between HSV-1 gC and rabbit hyperimmune antisera to both HSV-1 and HSV-2. Electrophoretic analysis also revealed reactivity between the rabbit antisera and peptides of HSV-1 gC generated by partial digestion with trypsin. These findings indicate that HSV-1 gC has one or more cross-reactive or type-common determinants that are readily detected, and therefore, the presence of antibodies reacting with HSV-1 gC in sera may not necessarily be indicative of an earlier infection with HSV-1.

The Ordovician to early Upper Carboniferous sequence of the Harz Mountains is laid down in a palinspastically reconstructed cross section. The problem caused by the stratigraphic position and the regional distribution of olistostromes is the reason for a detailed description of the coarse-clastic resediments of the Pre-Flysch and Flysch Phase. Resediments of the Pre-Flysch Phase are represented by the so-called graywackes of the Lower Devonian, pebbly mudstones (Middle Devonian), resedimented Hercynian Limestones (Lower—Upper Devonian) and allodapic carbonates of the "Flinz” facies (Middle Devonian — Lower Carboniferous). The Flysch Phase is consisting of three graywacke units: Upper Devonian Südharz-/Selke graywacke, Tanne series including the Sieber graywacke (Lower Carboniferous II - ? III) and the graywacke of the Northwestern Harz Mountains (Lower Carboniferous III - Namurian A). The base of the Tanne series is characterized by a locally distributed wildflysch, in which olistostromes are intercalated. Tanne series and Sieber graywackes are time-equivalent to the Kamm Quartzite of the Acker-Bruchberg -System. The graywacke facies types are interpreted in the sense of R. G. Walker (1975).

Monoclonal antibodies were prepared against herpes simplex virus type 1 (strain 14012) by two immunization procedures. Procedure A utilized infectious virus propagated in mouse cells, and procedure B utilized mouse cells infected with herpes simplex virus in the presence of cycloheximide and harvested 1 h after removal of the inhibitor. A total of 52 monoclonal antibodies were obtained against 10 herpes simplex virus proteins, including four glycosylated proteins (a 110,000-molecular-weight protein, gB, gC, and gD) and six nonglycosylated proteins (a 68,000-molecular-weight protein, ICP 9, ICP 8, ICP 6, ICP 5, and the immediate-early ICP 4). The antibodies were assayed against herpes simplex virus types 1 and 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioimmunoprecipitates, immunofluorescence, and neutralization. Using the reagents prepared, we concluded that the 110,000-molecular-weight protein, gD, ICP 9, ICP 9, ICP 6, and the 68,000-molecular-weight protein express both type-specific and cross-reactive antigenic determinants. In contrast, nine antibodies against gB all cross-reacted with herpes simplex virus type 2, whereas eight antibodies to gC all reacted type specifically.

Nucleotide sequence and mRNA localization studies have allowed the prediction of the amino acid sequence of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC). We immunized a rabbit with a conjugate of bovine serum albumin and a synthetic peptide having the same sequence as that deduced for amino acids 128 through 139 of HSV-1 gC. A very similar amino acid sequence has been predicted to exist in the related product, herpes simplex virus type 2 (HSV-2) gC, which was formerly designated gF. Preparations of crude antiserum and immunoaffinity-purified antibodies were obtained and shown to react in enzyme-linked immunosorbent assays with purified HSV-1 gC and HSV-2 gC. Although these antibodies did not detectably immunoprecipitate proteins from radiolabeled infected cell extracts, they reacted with HSV-1 gC and HSV-2 gC that were electrophoretically transferred to nitrocellulose membranes from polyacrylamide gels. These results confirm that HSV-1 gC and HSV-2 gC are immunologically related and also define a specific portion of HSV-1 gC that is conserved.

We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.

Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants.

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.

A rapid and reproducible enzyme-linked immunosorbent assay (ELISA) is described for determining antibodies in human sera against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The sera were absorbed for 30 min with heterologous virus-infected-cell extracts to remove cross-reacting antibodies and then were applied to ELISA plates containing the target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD and HSV-2 glycoproteins gD and gF. The absorbance index, defined as the ratio of A414 generated by a serum sample absorbed with a heterologous virus-infected-cell extract versus the A414 of a serum sample absorbed with an uninfected-cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2. Results of the ELISA for detecting antibodies against HSV-2, when compared with results obtained for the same sera by the microneutralization test, showed an index of overall agreement of 91%. Results of the ELISA for detecting antibodies against HSV-1, when compared with microneutralization test results for sera negative for HSV-2 antibodies but positive for HSV antibodies by ELISA, showed an index of agreement of 99%.