CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus.

Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer 'tethered' to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.

Targeted delivery of antisense DNA in woodchuck hepatitis virus-infected woodchucks.

An asialoglycoprotein-based DNA delivery system containing an antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of woodchuck hepatitis virus (WHV) was prepared. Experimental woodchucks were inoculated neonatally with the woodchuck virus 23 weeks before initiating the study, and all animals subsequently developed hepatitis as evidenced by the presence of measurable levels of circulating viral DNA. Animals were injected intravenously (i.v.) with asialoorosomucoid (AsOR)-poly-L-lysine complexes containing 0.1 mg kg-1 antisense DNA for five consecutive days. Levels of surface antigen did not differ substantially between treated and control animals. However, intravenous administration of complexed antisense DNA significantly decreased viraemia, as shown by a five- to 10-fold decrease in circulating viral DNA 25 days post treatment. The decline lasted for at least 2 weeks, after which there was a gradual increase in DNA levels. Antisense DNA alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels. We conclude that antisense oligo DNA can be targeted to the liver in vivo, resulting in a substantial and prolonged decrease in viral DNA levels in WHV-infected woodchucks.

Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.

The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.

Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells.

HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91-99). These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9. In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame. This region lies within the putative helicase domain of the UL9 protein. The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines. These results confirm that the UL9 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed. We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct. Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus. Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.

cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals.

Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.

The herpes simplex virus type 1 alkaline nuclease is not essential for viral DNA synthesis: isolation and characterization of a lacZ insertion mutant.

Herpes simplex virus type 1 (HSV-1) encodes a novel enzyme activity, the alkaline nuclease, whose precise role in the viral replication cycle remains obscure. The alkaline nuclease gene corresponds to the UL12 open reading frame, which is predicted to encode a protein of 626 amino acid residues. We describe the isolation and characterization of a null mutant of the gene for the viral alkaline nuclease in which 917 bp from the N-terminal half of the gene (corresponding to residues 70 to 375) were deleted and replaced by the insertional mutagen ICP6::lacZ. The resulting mutant virus, AN-1, was propagated in helper cells (S22) which express the wild-type version of the alkaline nuclease gene. Mutant AN-1 growth in Vero cells is severely restricted, although small amounts of infectious virus are produced. On the other hand, wild-type levels of viral DNA and late viral proteins are expressed in virus AN-1-infected Vero cells. These results indicate that the HSV-1 alkaline nuclease gene product is not essential for viral DNA synthesis but may play a role in the processing or packaging of viral DNA into infectious virions. Possible roles in the viral infectious cycle will be discussed.

Herpes simplex virus type 1 DNA synthesis requires the product of the UL8 gene: isolation and characterization of an ICP6::lacZ insertion mutation.

We investigated the role of the herpes simplex virus type 1 UL8 gene product in viral DNA replication. First, we unambiguously fine mapped the mutation in tsS38 (complementation group 1-26) to an open reading frame, designated UL8, predicted to encode an 80-kilodalton protein. Previous studies indicated that tsS38 was capable of synthesizing low to moderate levels of viral DNA at the nonpermissive temperature (C. T. Chu, D. S. Parris, R. A. F. Dixon, F. E. Farber, and P. A. Schaffer, Virology 98:168-181, 1979); thus, it was not clear whether the UL8 gene product is essential for viral DNA synthesis. Therefore, a deletion-insertion mutation was constructed in the UL8 gene by removing most of its coding sequences and replacing them with the Escherichia coli lacZ gene under control of the viral ICP6 regulatory signals. The resulting recombinant, hr80, was propagated in helper cells (S22) which express the wild-type version of the UL8 gene, but was incapable of forming plaques in Vero cells. Furthermore, hr80 was totally defective in the synthesis of viral DNA and late proteins under nonpermissive growth conditions. These results demonstrated that the UL8 gene product is essential for viral DNA synthesis.

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.

Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA.

Several HSV-1 proteins including the major capsid protein (VP5), two minor capsid proteins (VP11-12 and VP18.8), the alkaline nuclease and glycoprotein gH have been reported to be encoded by the left-most one-third of HSV-1 UL DNA. In this paper, we present physical mapping data and phenotypic analysis of six ts mutants whose mutations lie within this region and which collectively represent four functional complementation groups (1-6, 1-7, 1-10, and 1-26). In this study, mutants in complementation group 1-10 were found to be defective in the synthesis of viral DNA, late viral polypeptides, and the formation of mature capsid-like structures--properties characteristic of other ts mutants defective in functions required for viral DNA synthesis. Two DNA-positive mutants in complementation group 1-7 fail to induce capsid formation and probably possess mutations in coding sequences for VP5. Mutants in two other complementation groups (1-6 and 1-26) synthesize significant levels of viral DNA, late polypeptides, and capsids. The functions of the gene products represented by these mutants remain to be determined.