Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells

Abstract

Defective herpes simplex virus type 1 genomes are composed of head-to-tail tandem repeats of small regions of the nondefective genome. Monomeric repeat units of class I defective herpes simplex virus genomes were cloned into bacterial plasmids. The repeat units functioned as replicons since both viral and convalently linked bacterial plasmid DNA replicated (with the help of DNA from nondefective virus) when transfected into rabbit skin cells. Recombinant plasmids were packaged into virions and were propagated from culture to culture by infection with progeny virus. Replication was evidently by a rolling circle mechanism since plasmid DNA was present in a high-molecular-weight form in transfected cells. Circular recombinant plasmid DNA replicated with a high degree of fidelity. In contrast, linear plasmid DNA underwent extensive deletions of both viral and bacterial sequences when transfected into rabbit skin cells. Derivative plasmids, a fraction of the size of the parental plasmid, were rescued by transforming Escherichia coli with DNA from the transfected rabbit skin cells. These plasmids functioned as shuttle vectors since they replicated faithfully in both eucaryotic and procaryotic cells.

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus

“…Two populations of PrV DIPs [PrV(1) and PrV(2)] were generated by independent serial passage at high multiplicity of the standard PrV(Ka) strain in RK cells (4). Populations of DIPs at various levels of passage were used, starting with passage 33 [PrV (1) Media and solutions. The following media were used: Eagle synthetic medium (9) plus 5% dialyzed calf serum (EDS); EDS without P04 (EDS-PO4); EDS-PO4 plus 20 ,ug of 5-fluorouracil (FU) and 5 F.g of thymidine per ml (EDS-P04+FU); EDS-PO4 with 20 ,ug of FU, 10 ,ug of bromodeoxyuridine (BUdR), and 5 ,ug of deoxycytidine (CdR) per ml (BUdR medium); self-digested (nuclease-free) pronase (2 mg/ml) in 0.02 M NaCl-0.02 M Tris (pH 7.3) (pronase solution); and 0.15 M NaCl-0.015 M sodium citrate (pH 7.2) plus 4% sodium lauryl sarkosinate-97 (lx SSC-4% sarkosyl).…”

“…Since populations of DIPs becomne enriched for virions whose genomes probably can replicate and be encapsidated, common features of these repeated sequences may, in principle, include origins of replication, as well as recognition sites for cleavage and encapsidation. Indeed, it has been established that the DIPs of herpes simplex virus (HSV) contain genomes composed of reiterated segments of DNA that include an origin of replication, as well as the signals required for efficient cleavage-encapsidation (1,26,27,29). Because these DIPs have a clear replicative advantage, cyclical increases and decreases in the level of infectious virus are observed as the populations of DIPs are passaged at high multiplicity.…”

“…Coinfection of cells with this population of DIPs and helper virus causes the overproduction of some other viral proteins as well (23). Infection of cells with the other population of DIPs, [PrV (1)], on the other hand, does not detectably affect viral protein synthesis (23). Nevertheless, the biological properties of the two populations of DIPs appear to be quite similar (4).…”

Sequences within the Herpesvirus-Conserved pac 1 and pac 2 Motifs Are Required for Cleavage and Packaging of the Murine Cytomegalovirus Genome

“…Studies using HSV-1 amplicons, which consist of small tandemly repeated subgenomic segments containing an a sequence linked to a viral origin of replication, have defined the role of a sequences in cleavage and packaging (7,15,17,49,51). The importance of the a sequence in cleavage and packaging was demonstrated by the observation that DNA replication occurred following transfection of HSV-1 origin-containing plasmids into HSV-1-infected cells, but that concatemeric plasmid DNA was not cleaved or packaged unless ba, ca (51), or a sequences alone (15) were included in the plasmid.…”

Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences

“…We have previously reported (8, 31, 34) the initial characterization of cloning-amplifying defective virus vectors (amplicons) derived from herpes simplex virus (HSV). HSV amplicons can be constructed with any one of the three known replication origins of the HSV genome (1,7,17,(31)(32)(33). In addition, a functional HSV amplicon must contain a cleavage-packaging signal derived from the end of the S component or from the junction of the S and L components (reviewed in reference 27) of standard HSV DNA (35; R. R. Spaete and N. Frenkel, submitted for publication).…”