Cell, Vol. 54, 369-381, July 29, 1966, Copyright 0 1988 by Cell Press Inversion Events in the HSV-1 Genome Are Directly Mediated by the Viral DNA Replication Machinery and Lack Sequence Specificity Peter C. Weber,’ Mark D. Challberg,.t Nancy J. Nelson,t Myron Levine,* and Joseph C. Gloriaso’ *Department of Microbiology and Immunology and Unit for Laboratory Animal Medicine *Department of Human Genetics University of Michigan Medical School Ann Arbor, Michigan 48109 t Laboratory of Viral Diseases National Institute of Allergy and Infectious Diseases 9000 Rockville Pike Bethesda, Maryland 20892 Summary The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals in- volved in the inversion process. Several observations suggested an intimate association of the recombina- tion mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site- specific process, but rather is the result of generalized recombination mediated by the complex of gene prod- ucts that replicate the viral DNA. Introduction The genome of herpes simplex virus type 1 (HSV-1) is a 152 kb linear double-stranded DNA molecule that consists of a 108 kb long (L) and a 13 kb short(S) component. The L and S components are each flanked by the inverted re- peat sequences b (9 kb) and c (6.5 kb), respectively, and are both bracketed by the inverted repeat sequence a (250-500 bp), as shown in the following arrangement: (Sheldrick and Berthelot, 1974; Wadsworth et al., 1975; Hayward et al., 1975). The L and S components of the HSV-1 genome have been demonstrated to invert relative to each other, result- ing in the formation of four equimolar isomeric popula- tions of viral DNA (Hayward et al., 1975; Delius and Cle- ments, 1976). It has been suggested that these inversions are due to homologous recombination between the in- verted repeats flanking the L and S components (Shel- drick and Berthelot, 1974; Smiley et al., 1981). Later studies using engineered duplications of viral restriction fragments have implicated the a sequence as the cis- acting element through which a site-specific recombina- tion event occurs (Mocarski et al., 1980; Mocarski and Roizman, 1981,1982b; Chou and Roizman, 1985). The ex- istence of a virally encoded, transacting function that mediates the inversion process was inferred by the inabil- ity of plasmid-borne a sequences to undergo recombina- tion without HSV-1 superinfection (Mocarski and Roiz- man, 1982a). However, little is known about the molecular mechanism of this recombination event or the putative vi- ral functions involved. The bacterial transposable element Tn5, which consists of two inverted 1.5 kb IS50 insertion elements flanking a 2.8 kb central unique region containing a kanamycinlne- omycin resistance determinant, has been widely used for insertion mutagenesis in prokaryotes (Berg and Berg, 1983; deBruijn and Lupski, 1984). Recently, we have de- veloped a rapid means of identifying HSV-1 genes that are nonessential for replication in cell culture, using this trans- poson. Cloned HSV-1 restriction fragments containing Tn5 insertions in three S component genes were recombined into the viral genome without impairment of virus produc- tion in cell culture (Weber et al., 1987). In this report, we show that copies of Tn5 inserted into the HSV-1 genome undergo sequence inversion via high-frequency recombi- nation between the duplicated IS50 elements of the trans- poson. The cis- and trans-acting functions required for Tn5 inversion were systematically analyzed in transient ex- pression assays. The results of these studies indicate that the HSV-1 DNA replication machinery itself directs recom- bination between duplicated segments within the viral ge- nome in a manner that is independent of specific DNA se- quences. These findings strongly suggest that a similar mechanism underlies L and S component inversion in the HSV-1 genome, and have led to the reinterpretation of results from earlier studies concerning the role of the a se- quence in genome isomerization. Results Tn5 Undergoes Sequence Inversion when Inserted into the HSV-1 Genome HSV-1 (strain KOS) viruses containing Tn5 insertions within the US2, US4, and US5 genes were constructed in a previous study (Weber et al., 1987). During the charac- terization of these mutant viral genomes by restriction en- donuclease and Southern blot analyses, it was observed that certain restriction digests yielded unexpected frag- ments. The analysis of one of these genomes, US2::Tn5, is presented in Figure 1. Restriction enzymes that cleave within the inverted IS50 repeats of Tn5 (Pstl or Hindlll) yielded the expected restriction fragments, while enzymes that cleave asymmetrically within the central unique re- gion of the transposon (BarnHI or Sall) generated not only the predicted restriction fragments, but an equivalent