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Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; glIa, gIlb, and glIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; glll) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.

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Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (Us) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessaty for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-1) revealed the following. (i) None of the recombinant plaque isolates that were either TKor which had a deletion in the Us was virulent. (ii) Not all recombinant plaque isolates which were both TK+ and had an

The rate of accumulation of cytomegalovirus transcripts in permissively infected human embryonic lung (HEL) cells was analyzed at various times after infection by hybridization of infected cell RNA to undigested or restriction endonuclease-digested cytomegalovirus DNA fixed to nitrocellulose filters. Differences in patterns of transcript accumulation were determined by measuring the abundance levels of RNA which hybridized to different HindIII-, XbaI-, or EcoRI-generated fragments of cytomegalovirus DNA. The results showed that a small but significant amount of cytomegalovirus RNA was detectable within the first 3 h after infection and that the rate of accumulation of these transcripts was static during the first 24 h, but increased thereafter. In general, the viral transcripts accumulating in infected cells could be divided into three classes. Immediate-early RNA (synthesized in the absence of protein synthesis in infected cells) hybridizes predominantly to a very restricted part of the genome and can be identified during the first 2 to 4 h postinfection. Early RNA (synthesized up to about 24 h after infection) originates from most regions of the genome but is characterized by the presence of transcripts which hybridize in great abundance to certain fragments. Late RNA (synthesized after 24 h, i.e., after the onset of viral DNA synthesis) hybridizes in approximately equal abundance to most regions of the viral genome. These results showed that a block in the transition from immediate-early to early RNA did not account for the extended period of time that elapses between the time of infection and the initiation of viral DNA synthesis. Interestingly, despite rapid adsorption and penetration and a static level of accumulation of transcripts in the cultures during the first 24 h, the number of cells that synthesized detectable amounts of viral antigens increased steadily during this time.

that the Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome-a deletion that is related to the absence of virus virulence. This strain is, however, also defective in other genes involved in virulence. We show here that virulence can be restored by marker rescue of the Bartha strain to which an intact Us has been restored (but not to the parental Bartha strain) by sequences derived from approximate map units 0.460 and 0.505 of the wild-type virus genome. No difference in the ability to grow in cell culture was observed between parental Bartha, Bartha 43/25a (Bartha to which an intact Us has been restored), or the doubly rescued Bartha strains. However, only the doubly rescued Bartha strain was virulent for both chickens and pigs and replicated to high titers when inoculated directly into the brains of chickens. The sequences that could restore virulence to the Bartha 43/25a strain encode four genes, all of which are involved in processes leading to the assembly of nucleocapsids. Since these sequences rescue virulence, it appears that a function that plays a role in nucleocapsid assembly is defective in the Bartha strain and that this defect contributes to the lack of virulence of this virus.

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