The therapeutic potential of intracellular antibodies.

Antibody engineering techniques permit the exploitation of the highly specific and diverse nature of the humoral immune system. Intracellular antibodies, consisting minimally of the antigen-binding domains of the parent immunoglobulin protein, may be used to manipulate microbial and cellular target molecules. Applications of this technology described to date include inhibition of replication of an infectious virus, downregulation of growth factor receptors, and inactivation of oncoproteins in cell culture systems. The ability to functionally manipulate virtually any target molecule using intracellular antibodies is clearly beneficial to basic science and molecular medicine.

Identification and analysis of the simian varicella virus thymidine kinase gene.

The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.

DNA sequence of the simian varicella virus (SVV) gH gene and analysis of the SVV and varicella zoster virus gH transcripts.

The varicella zoster virus (VZV) glycoprotein H (gH) stimulates VZV-specific immune responses and may be involved in virus penetration. This study reports the genomic map position and the DNA sequence of a simian varicella virus (SVV) homologue of the VZV gH gene. A 32P-labeled VZV gH-specific DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment. The DNA sequence of the SVV HindIII B subclone was determined and analysis indicated a SVV open reading frame (ORF) homologous to several herpesvirus gH genes. The SVV gH ORF is 2559 base pairs in size and encodes a 852-amino acid protein. The SVV gH contains characteristics of a transmembrane glycoprotein including: 9 consensus N-linked glycosylation sites, a potential amino terminal signal sequence, and a predicted transmembrane segment located near the carboxyl terminus. The SVV and VZV gH genes exhibit 60.0% identity and the predicted polypeptides exhibit 54.5% identity. The SVV and VZV gH transcripts were analyzed and the promoter regions were compared. 32P-labeled SVV and VZV gH-specific DNA probes each hybridized to a single 2.9 kilobase transcript. The mRNA start sites of the SVV and VZV gH genes were determined by primer extension analysis, and alignment of the promoter regions indicated similar content and arrangement. The extensive conservation of SVV and VZV genes and predicted polypeptides further supports the use of SVV infection of non-human primates as a model of VZV infection of humans.

DNA sequence and transcriptional analysis of the simian varicella virus glycoprotein B gene.

The varicella-zoster virus (VZV) glycoprotein B (gB) is a major viral antigen which elicits immunity and neutralizing antibodies. In this study, the genomic map position and DNA sequence of a simian varicella virus (SVV) homologue of the VZV gB gene was identified and the transcript analysed. A 32P-labelled VZV gB DNA probe hybridized to a subclone of the SVV BamHI B restriction endonuclease fragment indicating the fine map position of SVV DNA sequences homologous to the VZV gB gene. The SVV gB DNA sequence was determined and analysis revealed a 2751 base pair open reading frame (ORF) with 71.1% identity to the VZV gB gene and 53.8% identity to the herpes simplex type 1 gB gene. The SVV gB ORF encodes a 916 amino acid polypeptide with a predicted molecular mass of 104K. The deduced SVV and VZV gB polypeptides share 78.9% amino acid identity and predicted N-linked glycosylation sites, cleavage sites and transmembrane regions. 32P-labelled SVV gB DNA and RNA probes hybridized to a 3.5 kilobase SVV polyadenylated transcript. Primer extension experiments identified transcript start sites for the SVV and VZV gB genes and permitted a comparison of the sequences upstream of the SVV and VZV gB ORFs. The SVV and VZV gB promoter elements are similar in content and align closely. The VZV gB transcript start site suggests a gB polypeptide initiation site which is inconsistent with the previously reported ATG start codon.

Characterization and mapping of simian varicella virus transcripts.

The size and genomic location of viral transcripts expressed in simian varicella virus (SVV)-infected Vero cells were determined. Total cellular RNA and polyadenylated RNA were isolated from SVV-infected and mock-infected Vero cells. Viral transcripts were detected by Northern blot hybridization analysis using overlapping SVV DNA probes representative of the entire SVV genome. The results indicated that all regions of the SVV genome are transcribed during SVV infection in vitro. At least 53 distinct viral RNA species ranging in size from 9.2 to 0.8 kb were detected. DNA probes derived from the SVV DNA long (L) and short (S) components hybridized to 44 RNAs (9.2 to 0.8 kb) and nine RNAs (4.9 to 0.8 kb), respectively. A transcript map of the SVV genome was constructed. The comparison made between the transcript maps of SVV and varicella-zoster virus (VZV) provides further support that the SVV and VZV genomes have an analogous gene organization.

The genomes of simian varicella virus and varicella zoster virus are colinear.

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.