Isolation and zonal fractionation of metaphase chromosomes from human diploid cells.

A simple method for isolating large quantities of human metaphase chromosomes has been developed. Fractionation of chromosomes from peripheral lymphocytes on a large scale is accomplished by velocity sedimentation in an A-XII zonal rotor.

JC virus enhancer-promoter active in human brain cells.

A human papovavirus, JCV, is the etiologic agent of the fatal demyelinating disease, progressive multifocal leukoencephalopathy. The JCV 98-base-pair tandem repeats, located to the late side of the viral replication origin, were shown to be a transcriptional regulatory element with enhancer-like activity in human fetal glial cells. These tandem repeats share significant homology with the 82-nucleotide rat brain-specific identifier RNA sequence.

The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4.

Human immunodeficiency virus type 1 (HIV-1) selectively infects cells expressing the CD4 molecule, resulting in substantial quantitative and qualitative defects in CD4+ T lymphocyte function in patients with acquired immunodeficiency syndrome (AIDS). However, only a very small number of cells in the peripheral blood of HIV-1-infected individuals are expressing virus at any given time. Previous studies have demonstrated that in vitro infection of CD4+ T cells with HIV-1 results in downregulation of CD4 expression such that CD4 protein is no longer detectable on the surface of the infected cells. In the present study, highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) from AIDS patients were obtained and purified by fluorescence-automated cell sorting. They were examined with the methodologies of virus isolation by limiting dilution analysis, in situ hybridization, immunofluorescence, and gene amplification. Within PBMCs, HIV-1 was expressed in vivo predominantly in the T cell subpopulation which, in contrast to the in vitro observations, continued to express CD4. The precursor frequency of these HIV-1-expressing cells was about 1/1000 CD4+ T cells. The CD4+ T cell population contained HIV-1 DNA in all HIV-1-infected individuals studied and the frequency in AIDS patients was at least 1/100 cells. This high level of infection may be the primary cause for the progressive decline in number and function of CD4+ T cells in patients with AIDS.

Characterization of a surrogate TATA box promoter that regulates in vitro transcription of the simian virus 40 major late gene.

The presence of a surrogate TATA box sequence located ca. 30 nucleotides upstream of the major late RNA start site at nucleotide position (np) 325 (Brady et al., Cell 31:625-633, 1982) has been confirmed, and its structural specificity has been determined by the generation of additional base substitution mutations at the KpnI restriction site (np 294) in cloned simian virus 40 DNA. Two mutants generated new RNA initiation sites upstream of the np 325 start site and continued to utilize the authentic start site, but with decreased efficiency. The replacement of either one or both cytosines by thymines at np 298 and np 299 specifically enhanced in vitro transcription from the np 325 start site by 430 and 800%, respectively. This enhancement was due to conversion of the simian virus 40 late promoter present in the wild-type virus to a sequence that is similar to the TATA box present in the simian virus 40 early promoter.

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro.

We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

Simian virus 40 early promoter mutations that affect promoter function and autoregulation by large T antigen.

A set of nine mutants containing point mutations, and small deletions or insertions, were constructed in the early promoter region of simian virus 40 (SV40) to determine the role of the DNA sequences between the TATA box and the six upstream G + C-rich clusters in early transcription. The mutant templates were tested for transcription activity in vitro in HeLa cell extracts and in vivo in CV-1 and COS cells using the chloramphenicol acetyl transferase gene (CAT) assay. Both in vitro and in vivo results show that the narrow region from nucleotide positions 38 to 41 is an important domain of the early promoter. Deletion and insertion mutations most strongly affect the level of transcription. Specifically a four base-pair deletion in the promoter region enhances the level of transcription four- to sixfold in vitro, but causes a fourfold suppression of CAT gene expression in the in vivo assay. These opposite effects may result from changes in spacing under in vitro and in vivo conditions between the TATA box and the G + C-rich motifs where transcription factors may make simultaneous contact. Of the three T antigen binding sites (I, II and III), sites I and II have already been shown to be involved in the autoregulation of early transcription. Our mutational analyses demonstrate the role of site III, which partially overlaps with nucleotide positions 38 to 41, in the autoregulation of the SV40 early promoter.

Envelope-specific antibodies in the saliva of individuals vaccinated with recombinant HIV-1 gp160.

HIV-1-specific antibodies have been detected in the saliva of seropositive individuals and may play a role in preventing oral transmission of the virus. We have analyzed saliva samples obtained from HIV-1-seronegative individuals who were immunized with various dosages of a recombinant HIV-1 envelope glycoprotein (gp160) vaccine for the presence of antibodies to HIV-1. Antibodies specific for envelope glycoproteins were detected in saliva from all of the volunteers, with those vaccinated with the higher doses of 640 and 1,280 micrograms showing the strongest responses. Peak salivary antibody titers were obtained 4-14 weeks after vaccination; they then gradually dropped in parallel with serum antibody titers. These envelope-specific antibodies were detected in whole saliva and in submandibular saliva but not in parotid saliva, suggesting that the source of antibodies in saliva is from serum transudation. The class of reactive antibodies was found to be IgG. The HIV-1-specific antibodies in the saliva of vaccinated individuals may offer local protection against HIV-1 infection.

Isolation of HIV-1 from plasma of infected individuals: an analysis of experimental conditions affecting successful virus propagation.

Experimental conditions affecting the successful propagation of HIV-1 from the plasma of seropositive individuals were examined. It was determined that whole blood samples collected with lithium heparin as the anticoagulant, immediate plasma separation, and immediate culturing were best suited for obtaining viable virus from plasma. Virus was isolated by infecting fresh phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cells (PBMCs) with plasma followed by weekly cocultivation with new target cells. The plasma virus isolation rate was the greatest and HIV-1 titers were the highest for those individuals with less than 200 CD4+ cells/mm3 and decreased as the level of CD4+ cells approached normal values. We were able to obtain positive cultures from 29.5% of those patients with CD4+ counts greater than 500/mm3. HIV-1 titers in plasma also correlated with high serum p24 antigen levels when serum was treated with glycine to dissociate antigen-antibody complexes.

Suppression of HIV-1 reverse transcriptase activity by mycoplasma contamination of cell cultures.

The detection of HIV-1 in human peripheral blood lymphocytes is routinely carried out by cocultivation of test cells with normal peripheral blood mononuclear cells (PBMC). The presence of virus is evidenced by cytologic observation of syncytia or by detecting viral reverse transcriptase (RT) and/or p24 antigen in the culture supernatant fluid. Syncytia formation is almost always associated with the presence of virus as measured by RT, although many RT-positive cultures do not form syncytia. As part of a large screening program, we identified three cultures that showed syncytia but were RT negative. The basis for these discrepant observations was contamination of cultures with mycoplasma that interfered with the RT assay and thereby obscured virus detection. Treatment of cultures with BM-cycline removed mycoplasma contamination and restored RT activity. The present findings indicate the need for caution in the interpretation of negative RT results during HIV-1 isolation and especially in cultures that show evidence of syncytia formation.

Sequential formation of vaccinia virus proteins and viral deoxyribonucleic acid replication.

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of "early viral protein" which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The "early" and "late" proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of "early" protein is discussed.

Sequential protein synthesis following vaccinia virus infection.

Inhibition of HeLa cell protein synthesis and the sequential synthesis of viral proteins were followed by pulse-labeling infected cells with (14)C-phenylalanine. Proteins were resolved by polyacrylamide gel electrophoresis. The viral origin of native proteins was confirmed by immunodiffusion. The inhibition of host protein synthesis and the synthesis of early viral proteins occur 1 to 3 hr after infection. This early sequence of events also occurs in the presence of 5-fluorodeoxyuridine, an inhibitor of deoxyribonucleic acid synthesis. Other viral proteins are synthesized at a later time. Those proteins which are not made in the absence of viral deoxyribonucleic acid synthesis can be further subdivided into intermediate and late classes. The intermediate protein is synthesized before the late proteins but does not appear to be a precursor of them. Many more viral polypeptides were resolved by polyacrylamide gel electrophoresis after solubilization of the entire cytoplasmic fraction with sodium dodecyl sulfate. Virion and nonvirion proteins were identified. Kinetic experiments suggested that certain structural proteins as well as certain nonstructural proteins are made early, whereas others of both classes are made primarily at later times.

Inhibition in the joining of DNA intermediates to growing simian virus 40 chains.

Viral DNA synthesis was inhibited for 1 h by the addition of 5-fluorodeoxyuridine (FUdR) to simian virus 40 (SV40)-infected cultures at 28 to 30 h postinfection. The subsequent addition of (3)H-thymidine to the inhibited cultures reverses the effect of the inhibitor, and during a 1-min labeling period there is rapid synthesis of SV40 DNA. By alkaline sedimentation analysis, it is observed that in FUdR-treated cultures there is synthesis of 4S SV40 DNA intermediates but there is a block in the joining of these intermediates to growing SV40 chain cultures. In addition to 4S fragments that are associated with replicating SV40 molecules, there is accumulation of SV40 DNA in the 6 to 8S region which is observed in neutral sucrose gradients. In an inhibited culture that is pulsed for 1 min with (3)H-thymidine and then chased for 10 min, accumulation of a Component II (Comp. II)-like material is observed. This Comp. II has the same neutral sedimentation characteristics and yields the same R(I) restriction endonuclease product as does authentic Comp. II. However, in alkali it is seen that it is composed of fragmented SV40 DNA. The basis for the failure of 4S fragments to join to growing SV40 chains is discussed. A model in which there is a requirement for two DNA polymerases and a ligase to permit SV40 DNA chain growth is proposed which is consistent with the data presented.