SummaryThe Rhs family comprises a set of composite elements found in the chromosomes of many natural Escherichia coii strains.
Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting ofhead-to-tail reiterations ofsequences derived from the end ofthe S component ofthe standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to M 100 X 106 DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.The standard genome of herpes simplex virus type 1 (HSV-1) is a linear, double-stranded DNA molecule with Mr 100 X 106 (1, 2). Topologically, the standard HSV DNA is terminally reiterated and consists oftwo covalently linked components, L and S, each of which contains a stretch of unique viral DNA sequences surrounded by inverted repeats: the unique sequences of L (UL) surrounded by the ab and b'a' inverted repeat sequences, and the unique sequences ofS (Us) bounded by the ac and c'a' inverted repeats (3-6). The L and S components have been shown to invert relative to each other, yielding equimolar amounts of four isomeric structures. (3,(5)(6)(7)(8).Whereas recently plaque-purified virus stocks predominantly contain standard HSV genomes, defective virus populations obtained through serial undiluted propagation of such stocks have been shown to contain variable proportions of variant DNA molecules in which greater than 90% of
Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets ofthe standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (Mr 100 X 106). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in Mr from 5.5 to 100 X 106. These smaller DNA species were shown to consist of different integral numbers (from I to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (i) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (ii) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (iii) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm.The standard DNA genomes of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are approximately 100 X 106 in Mr and are organized in two covalently linked components, L and S (1-3). The L component consists of unique sequences UL (Mr, 67 X 106) bounded by the inverted repeats ab and b'a' (Mr 5.8 X 106). The S component consists of unique sequences Us (Mr, 9 X 106) bounded by the inverted repeats ac and c'a' (Mr, 4.1 X 106). The sequence a is present in one or few copies at the L and S termini and at the L-S junctions (3-8). The L and S components invert relative to each other, forming four equimolar isomers (3, 9-11).In addition to the standard (helper) virus genomes, serially passaged virus stocks also contain variable proportions of defective virus genomes consisting of tandem reiterations of sequences oflimited complexity (repeat units). All ofthe defective HSV genomes characterized to date contain, within their repeat units, DNA sequences derived from the end ofthe S component of standard virus DNA. However, they fall into two distinct classes on the basis of the additional sequences that they...
The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.
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