Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.     

Analysis of temperature-sensitive mutations in the simian virus 40 gene encoding virion protein 1.

Temperature-sensitive (ts) assembly mutants of the tumorigenic virus simian virus 40 (SV40) fail to follow the normal pathway of virion morphogenesis at 40 degrees C. The mutations were previously mapped to the gene coding for the major virion protein VP1 and fall into three groups: tsB, tsBC, and tsC. We have determined the tsB/C mutations by DNA sequence analysis and deduced the corresponding amino acid substitutions. We find that the mutations are global and span 68% of the VP1 gene. They result predominantly in single amino acid substitutions. The B mutations are localized between nucleotides 1667 and 2091, spanning the VP1 amino acid residues 54-195. With the exception of one mutation in tsC260, the C group mutations occur between the nucleotides 2141 and 2262, spanning VP1 residues 212-252. The tsBC substitutions are not localized within a distinct region. We present a model for the VP1 structure. The model correlates the distribution of ts assembly mutations in the SV40 VP1 gene with the VP1 functional domains, deduced form the phenotypes exhibited by the assembly mutants, and the VP1 structural domains, deduced recently from the cryoelectron microscopic studies of the SV40 virions. We summarize the behavior of the SV40 ts mutants and discuss the possible relationship between the ts phenotype and amino acid substitutions.     

Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product.

Formaldehyde fixation of simian virus 40 (SV40)-infected CV-1 cells at appropriate times after infection permits us to isolate crosslinked complexes of SV40 minichromosomes during the time of DNA replication and during packaging with viral proteins. Such crosslinked complexes can be separated on the basis of density on CsCl/guanidine . HCl density gradients. During the course of these studies we observed the presence of a low molecular weight protein in a region of the gradient much enriched with viral nucleoproteins. This protein is present only in infected cells and has a molecular weight and amino acid composition consistent with it being the product of the so-called SV40 agnogene.     

Rapid turnover of acetyl groups in the four core histones of simian virus 40 minichromosomes.

The four core histones (H2a, H2b, H3, and H4) bound to simian virus 40 minichromosomes isolated from infected cells contain rapidly labeled acetyl groups in internal positions of the histone polypeptide chain. Upon chase, these acetyl residues decay with a half-life of less than 15 min. The acetyl groups are incorporated in histones bound to mature chromosomes and not in newly synthesized histones bound to replicating viral chromosomes. The rate of acetate incorporation is not related to the degree of steady state acetylation of the individual viral or cellular histones. This rate is 4-fold higher for the viral chromatin than for its cellular counterpart isolated from the same nuclei. The possible role for histone acetylation in viral genome expression is discussed.     

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.     

Thermolabile T (tumor) antigen from cells transformed by a temperature-sensitive mutant of simian virus 40.

Partially purified tumor (T) antigen from a strain of Chinese hamster lung cells transformed by wild-type simian virus 40 (SV 40) and either of two temperature-sensitive SV 40 mutants has been studied as a DNA binding protein. The DNA binding activity present in the T-antigen-containing fractions is inhibited by purified hamster anti-T IgG but not by equivalent amounts of nonimmune hamster IgG. T from either wild-type- or tsC219-transformed cells is relatively stable during heating at 44 degrees compared to T prepared from tsA239-transformed cells. These results strongly suggest that T is a product of the SV 40 A gene.     

Import of simian virus 40 virions through nuclear pore complexes.

How the DNA tumor virus, simian virus 40, reaches the nucleus is unknown. In this report we have tested the affinity of simian virus 40 toward the nucleus by microinjecting virion particles into the cytoplasm under conditions in which cell-surface-mediated viral infection was blocked. Subcellular localization of viral structural proteins Vp1, Vp2, and Vp3, large tumor antigen, and virion particles was followed immunocytochemically and ultrastructurally. Both virion particles and viral structural proteins localized in the nucleus within 1-2 hr after cytoplasmic injection and subsequently expressed large tumor antigen, which was detected in the nucleus as early as 3 hr after cytoplasmic injection. Vp1 and large tumor antigen nuclear accumulation, as well as virion nuclear entry, were blocked by wheat germ agglutinin and an anti-nucleoporin monoclonal antibody, mAb 414. Virion particles were visualized in the vicinity of nuclear pores and in the cytoplasm with this agent. We conclude that virion particles are karyophilic and enter through nuclear pores. This study suggests that virion structural proteins facilitate virion import into the nucleus and viral gene expression.     

Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells.

Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition of appropriate linker oligonucleotides. Fragments originating from each of the two isomeric forms of EHV-1 DNA were contained in this library of clones. Supramolar DNA fragments present only in the DNA of defective interfering (DI) particles of EHV-1 were generated from Bgl II digestion of DNA preparations enriched for EHV-1 DI particles and were cloned as Bgl II and EcoRI fragments into the plasmid vector. The cloned viral sequences represented in this defective genome mapped to the S region of EHV-1 DNA. Blot-hybridization analysis of EHV-1 transformed and tumor cell DNAs with the cloned BamHI B fragment confirmed that subgenomic viral sequences are present and indicated that those sequences map to the viral genome between 0.32 and 0.43 map unit.     

Discovery of functional noncoding elements by digital analysis of chromatin structure

We developed a quantitative methodology, digital analysis of chromatin structure (DACS), for high-throughput, automated mapping of DNase I-hypersensitive sites and associated cis-regulatory sequences in the human and other complex genomes. We used 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 tags from erythroid cells. Tags were mapped to the human genome, and a quantitative algorithm was applied to discriminate statistically significant clusters of independent DNase I cutting events. We show that such clusters identify both known regulatory sequences and previously unrecognized functional elements across the genome. We used in silico simulation to demonstrate that DACS is capable of efficient and accurate localization of the majority of DNase I-hypersensitive sites in the human genome without requiring an independent validation step. A unique feature of DACS is that it permits unbiased evaluation of the chromatin state of regulatory sequences from widely separated genomic loci. We found surprisingly large differences in the accessibility of distant regulatory sequences, suggesting the existence of a hierarchy of nuclear organization that escapes detection by conventional chromatin assays.     

Nucleosome segregation at a defined mammalian chromosomal site.

When animal cells replicate chromatin under conditions precluding new histone biosynthesis, half of the daughter DNAs are devoid of nucleosomes and are sensitive to staphylococcal nuclease. DNA sequences resistant to nuclease are associated with preexisting nucleosomes, which redistribute to progeny DNA duplexes during replication. We labeled newly replicated DNA sequences in a simian virus 40 (SV40)-transformed Chinese hamster cell clone with 5-bromodeoxyuridine (BrdUrd) in the presence and absence of a protein biosynthesis inhibitor, emetine. We resolved single-stranded BrdUrd- and dT-DNA sequences protected from nuclease digestion by nucleosomes and determined from which strands of the integrated viral DNA parental template (dT) and newly replicated progeny (BrdUrd) sequences were derived. Because we knew that the cell clone studied contained all of its integrated SV40 DNA at a single chromosomal site, we were able to determine that preexisting nucleosomes segregated to only one of the two daughter duplexes containing the integrated viral sequence. Additionally, in the presence of emetine, the integrated viral origin of replication, ORIsv, appeared not to function as a chromosomal replication origin, perhaps reflecting the drug's effect on synthesis of SV40 large tumor antigen.     

Subnuclear systems for synthesis of simian virus 40 DNA in vitro.

We have developed two subnuclear systems for synthesis of DNA of simian virus 40 in vitro. We prepare chromatin from infected cells by the method of Hancock [(1974) J. Mol. Biol. 86, 649-663]; these "chromatin bodies" can be disrupted and large debris can be pelleted, leaving a supernatant ("soluble system"). Both chromatin bodies and the soluble system incorporate deoxyribonucleoside triphosphates into nucleoprotein complexes that contain simian virus 40 DNA. The DNA labeled in short pulses sediments in neutral sucrose gradients slightly faster than mature simian virus 40 DNA, as expected for replicating intermediate. When rebanded in alkaline sucrose gradients, about half of the radioactivity is found in short strands (200-300 nucleotides) and half in longer strands (up to full viral size). When these systems are supplemented with a cytoplasmic preparation from HeLa cells, synthesis is stimulated about 5-fold, and the short strands are converted into strands of up to full viral length as well as into covalently closed circles. These subnuclear DNA-replicating systems should be useful for biochemical fractionation and characterization of some of the proteins required for DNA replication.     

Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production

Human cells resist viral infections by a variety of mechanisms. Viruses must overcome host cell restrictions to successfully reproduce their genetic material. Here, we identify a host restriction to viral replication that acts at the stage of particle assembly. Viral protein U (Vpu) is an HIV-1 accessory protein that enhances particle assembly and release in most human cells, but not in simian cells. By using human-simian cell heterokaryons, we show that the inhibition of assembly in human cells is dominant. Vpu overcomes the block to assembly in human cells and in human-simian heterokaryons. The HIV-1 vpu gene may have evolved to counteract an assembly restriction that is present in human cells.