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Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1, lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element(s) was present in the LTR. For example, in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements (and no Sp1 binding sites), a hierarchy of cellular permissivity to virus replication (peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present.

The structure of polyoma virus replicative intermediates isolated from infected 3T6 cells was analyzed by two-dimensional agarose gel electrophoresis (Sundin and Varshavsky, Cell 21:103-114, 1980) and quantitative electron microscopy (Krauss and Benbow, J. Virol. 38:815-825, 1981). DNA replication was initiated at a single site (ori) in essentially all of the replicative intermediates. Most of the early replicative intermediates were formed by unidirectional synthesis in the direction of early transcription. Most mid- and late replicative intermediates contained two replication forks which had traveled unequal distances from the origin. Asynchronous initiation of the two growing forks was postulated to account for these observations.

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.

Nuclear matrix prepared from mouse 3T6 cells lytically infected with polyoma virus retained significant amounts of the 100K T antigen and intact viral genomes. Bound T antigen was resistant to the extraction by high salt (2 M NaCl), detergent (1% Triton X-100) and exhaustive DNAase treatment. Only conditions sufficient to disrupt the integrity of the matrix itself solubilized the matrix T antigen. During the time period of 16-30 hr after infection, both the accumulation (in microgram) and the incorporation of 35S-methionine into T antigen increased steadily in cell extracts to a peak at 26 hr and then declined. In contrast, the amount of labeled T antigen retained by the matrix was relatively constant over the same time period. Matrix-bound T antigen was more highly phosphorylated and newly synthesized compared with the extractable T antigen. Viral DNA steadily accumulates in nuclei and on the matrix from 18 to 30 hr after infection. The fraction of viral DNA retained by the matrix was greatest early in infection (25% at 16 hr), declining to less than 10% by 24 hr. These data are consistent with the existence of a fixed (and limited) number of sites for T antigen (more highly phosphorylated) on the matrix and implicate the nuclear matrix as a site of viral DNA replication and possibly encapsidation.

Polyoma virus minichromosomes were isolated from infected 3T6 cells by hypotonic extraction of isolated nuclei. The kinetics of in vitro DNA synthesis in the nuclear extract was similar to that observed with intact nuclei. The majority of the products of in vitro DNA synthesis sedimented with replicative intermediate (RI) minichromosomes and migrated as two bands (RI-a and RI-b) on 1.4% agarose gels. The kinetics of deoxynucleotide monophosphate incorporation into these species was consistent with the existence of several rate-limiting steps in in vitro replication by polyoma minichromosomes. Electron microscope analysis showed that the RI-a band consisted almost entirely of RI theta structures ranging from 46 to 87% replicated, with one-half of all theta structures 67 +/- 4% replicated. The RI-b material was more complex, consisting of sigma and alpha structures with tails ranging from 7 to 114% of polyoma genome length and, less frequently, of linked and multiple linked dimeric structures.