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Intracellular forms of pseudorabies virus parental DNA were examined before and after the onset of viral DNA synthesis. Before initiation of synthesis, parental viral DNA acquires single-stranded ends. Circular and concatemeric molecules are also observed, indicating that the single-stranded ends are complementary. Viral DNA replication is initiated at an internal site within the DNA molecule, giving rise to characteristic replicative loops with single-stranded regions in the trans position. Such replicative loops were seen in unit-size (and smaller than unit-size) linear molecules as well as in circular and concatemeric molecules. These results show that the parental viral DNA molecules that acquire single-stranded ends, and consequently are able to form circles and concatemers, proceed to replicate.

The replication of PRV DNA occurs in two phases, early and late. During the early stages of infection newly synthesized DNA is associated with molecules sedimenting with an S-value up to two-fold greater than that of mature viral DNA. These molecules represent unit-size linear or circular molecules, as well as small concatemers in the process of replication. Initiation of replication occurs at a site situated 20 micron from one of the ends as well as at or near the end of the molecules. At later times, newly synthesized DNA is associated with large, "tangled" concatemers containing single-stranded segments of DNA. Our results indicate that at least some of the single-stranded DNA may be produced during the extraction procedure. Analysis of the large, "tangled" concatemers with restriction enzymes shows that they consist of linear arrays of viral DNA molecules.

The rate of pseudorabies virus DNA elongation was measured by three different techniques: density shift experiments, radioautography examined by light microscopy, and radioautography examined by electron microscopy. The rate of the fork movement at 37 degrees C was estimated to be approximately 1 micron/min.

The envelope of pseudorabies virions can be removed by treatment with Triton X-100, leaving the nucleocapsid intact. The viral envelope removed in this fashion contains all the glycoproteins and about 40 per cent of the proteins of the virions. Analysis by polyacrylamide gel electrophoresis revealed that the envelope proteins migrate as four distinct peaks, all of which contain glycoproteins. The proteins of the nucleocapsids migrate in the gels as six distinct peaks.