The DNAs of here sim lex virus (HSV) 1 and 2 consist of two components, L and S, each composed of unique sequences bracketed by inverted repeats. In this study we have probed the structure of the reiterated regionsof the S component in marker rescue experiments involving transfection of cells with mixtures of intact HSV-1 mutant viral DNA and individual DNA fragments generated by restriction endonuclease digestion of wild-type HSV-1 or HSV-2 DNAs. The results were as foflows: (i) HSV is diploid for the wild-type sequences that rescue two temperature-sensitive (ts) mutants. DNA fragments from both reiterated regions of theS component of HSV-1(F) DNA can rescue tsLB2 and tsD mutants. (ii) Identity of the entire reiterated sequence at both ends of S is not obligatory because only one end of the S component of wild 1)notype virus HSV-1(1061) rescues tsD even though both ends rescue tsLB2.(ill) Genes in both reiterated sequences can be expressed. We produced, by marker rescue experiments, recombinants with heterotypic ends of the S component, and these specified correspon in polypeptides characteristic of both HSV-1 and HSV2. (iv) The reiterated sequences of the S component may contain a region of obligatory identity. Thus, several recombinant clones produced by rescue with HSV-2 DNA contained identical HSV-2 DNA insertions within both reiterated regions of the HSV-1 S component. Consistent with this conclusion, the termini of the S component in the heterodiploids described in iii were identical by restriction enzyme analysis. (v) The observation that HSV DNA can be expanded by at least 5 X 100 by means of insertion in the S component suggests that it can be a vehicle for exogenous DNA. Herpes simplex virus 1 (HSV-1) specifies approximately 50 polypeptides which form at least three groups, designated a, fl, and y, whose synthesis is coordinately regulated and sequentially ordered (1). Its DNA (97 X 106 molecular weight) consists of two covalently linked components designated L and S. comprising 82 and 18% of total DNA, respectively (Fig. 1A). Both L and S consist of unique sequences (UL and Us) bracketed by inverted repeats (2). The reiterated sequences of L, designated ab and b'a', each consists of 6% of total DNA, whereas the reiterated sequences of S, designated a'c' and ca, each consists of 4.3% of total DNA (3). HSV-1 DNA extracted from virions consists of four equimolar populations differing in the orientation of the L and S components relative to each other (4, 5). The four populations have been designated P (prototype), IL (inversion of L), Is (inversion of S), and ISL (inversion of both S and L) (Fig. 1A). One consequence of this DNA arrangement is that restriction endonucleases that do not cleave within the reiterated regions generate three classes of fragments. The first class, corresponding in concentration to the molarity of intact DNA, comprises fragments mapping between the first and last cleavage within the unique L and unique S regions. The second class comprises the four terminal fragmen...

R325-lTK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the a22 gene and the wild-type (,) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of 0 proteins was delayed and the duration of synthesis of y proteins was extended in R325-PTK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (Y2) gene expression, a recombinant carrying the deletion in the a22 gene and a 'y2-TK gene (R325-Y2TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric y2-TK gene. In Vero cells, the Y2-TK gene of R325-y2TK was expressed earlier than and at the same level as the y2-TK gene of R3112. In the confluent resting HEL cells, the expression of the 'y2-TK gene of the a22-virus was grossly reduced relative to that of the at22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-,TK' virus was reduced relative to that of

SUMMARYEquine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of Mr 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of Mr 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent Mr of 108K (140K under nonreducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a l12K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

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SUMMARYFour intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interactingwith type specific neutralizing antibody. The analysis shows that each recombinant possesses a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type I and pseudorabies viruses.