Maurice Green

Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein

HIV-1 encodes a potent trans-activator protein, tat, which is essential for viral gene expression. To study tat domains that function in trans-activation, we chemically synthesized the 86 amino acid tat protein (tat-86) and tat mutant peptides. Remarkably, tat-86 is rapidly taken up by cells, and produces a massive and specific stimulation of HIV-LTR-driven RNA synthesis. Mutant peptides of 21 to 41 amino acids exhibit significant activity. Only two regions are essential for trans-activation; we suggest that one represents an activation region and the other, a nucleic acid binding or nuclear targeting region. Amino acid substitutions within these regions greatly reduce trans-activation, demonstrating the functional significance of these domains. The N-terminal 37 amino acids and exon 2 are not essential. Thus, tat is similar to regulatory proteins of Ad E1A and BPV1 E5 oncogenes, requiring only small domains for autonomous function.

Biochemical studies on adenovirus multiplication: IV. Isolation, purification, and chemical analysis of adenovirus

Abstract A method is described for the isolation and purification of type 2 adenovirus from infected suspension cultures of KB cells. Yields of 5–15 mg of purified virus were obtained from 6 to 9 × 10 8 cells. The procedure involved extraction with Tris buffer, treatment with genetron, centrifugation upon a layer of RbCl solution, and two RbCl-density gradient centrifugations. The virus was found to contain 13% DNA and 87% protein. Base analysis of viral DNA gave molar percentages of 22, 21, 27, and 29 for adenine, thymine, guanine, and cytosine, respectively. These ratios are strikingly different from that of host cell DNA in which adenine and thymine predominate. Evidence for the purity of the virus and for the double-stranded nature of viral DNA is discussed. Type 4 adenovirus prepared by the above procedure had a similar chemical composition and DNA base ratios as type 2 adenovirus.

Biochemical studies on adenovirus multiplication

Abstract Multiple infection of mass cultures of KB cells with type 2 adenovirus resulted in marked stimulation of incorporation of P32-orthophosphate into RNA mononucleotides and DNA. Studies at several time intervals revealed greatest incorporation of P32 into RNA at 9–18 hours, into DNA at both 9–18 and 18–27 hours after infection. The data are interpreted as indicating a virus-stimulated incorporation of medium P32 at 9–18 hours into the phosphate pool of the cell. At this time, the acid-soluble nucleotide and phospholipid fractions were also found to be more active in infected than in uninfected cells. The prolonged increased uptake of isotope into the DNA of the infected cell may reflect, in addition to a more highly labeled phosphate pool, a stimulated synthesis of a new DNA, perhaps virus precursor.

Mutational Analysis of HIV-1 Tat Minimal Domain Peptides: Identification of Trans-Dominant Mutants That Suppress HIV-LTR-Driven Gene Expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.

An adenovirus E1a protein region required for transformation and transcriptional repression

The adenovirus E1a region encodes two closely related gene products: 243 and 289 amino acid phosphoproteins. These proteins differ in their primary sequence only by 46 amino acids unique to the 289 amino acid protein. By constructing single-base substitution mutants we localized two functional regions of these E1a proteins: one required for efficient transcriptional activation, another required for efficient transcriptional repression. The 289 amino acid protein contains both regions and appears to function primarily as a transcriptional activator. The 243 amino acid protein lacks the transcriptional activation domain and appears to function primarily as a transcriptional repressor. Mutations within a highly conserved region define a novel class of transformation-defective mutants. These mutant E1a proteins can still efficiently activate transcription of early viral and cellular genes but cannot repress transcription of target genes. The fact that viral transformation may require a transcriptional repression function provides new insights into the mechanism by which adenovirus transforms cells.

Functional domains of adenovirus type 5 E1a proteins

Adenovirus E1a proteins function in transcriptional activation, transcriptional repression, cellular DNA synthesis induction, and cellular transformation. Here we examine the role of the previously undefined E1a region 1, the last of three conserved E1a regions to be characterized. Region 1 is required for transcriptional repression, transformation, and DNA synthesis induction, but not transcriptional activation. These results support our previous suggestion that transcriptional repression is the basis of E1a-mediated transformation. Two conserved regions (regions 1 and 2), present in both early E1a proteins, are essential for transcriptional repression, transformation, and induction of DNA synthesis. In contrast, mutagenesis suggests that transcriptional activation requires only 49 amino acids (region 3) unique to the 289 amino acid E1a protein. This we prove by demonstrating that a 49 amino acid region 3 synthetic peptide efficiently activates an E1a-inducible promoter. This peptide is the smallest known protein fragment functioning as a transcriptional activator.

Thirty-one human adenovirus serotypes (Ad1-Ad31) form five groups (A-E) based upon DNA genome homologies.

Abstract The DNA homology relationships among 31 human adenovirus serotypes (Ad1–Ad31) were investigated by liquid-phase molecular hybridization, using in vitro labeled viral DNA as probe. Hybridizations were carried to 40 times the C 0 t 1 2 and were assayed by batchwise chromatography on hydroxylapatite (HAP), and in some experiments by use of the more stringent S1 nuclease procedure. Five distinct DNA homology groups, A to E, were identified. DNAs of group A Ads (Ad12, 18, and 31) hybridized 48 to 69% with each other and 8 to 20% with DNAs of other serotypes (HAP). DNAs of group B Ads (Ad3, 7, 11, 14, 16, and 21) hybridized 89 to 94% (HAP; 81 to 89% by S1 nuclease) with each other and 9 to 20% (HAP; 8 to 15% by S1 nuclease) with DNAs of other types. DNAs of group C Ads (Ad1, 2, 5, and 6) hybridized 99 to 100% with each other and 10 to 16% with DNAs of other types (HAP). DNAs of group D Ads (Ad8–10, 13, 15, 17, 19, 20, and 22–30) hybridized 95 to 99% (HAP; 88 to 98% by S1 nuclease) with each other and 4 to 17% with DNAs of other types. Ad4 DNA hybridized to 4 to 23% (HAP; 3 to 22% by S1 nuclease) with DNAs of other types, and thus Ad4 is the only member of group E. Members within all groups except group A were closely related. Members within group A showed considerable heterology, and six isolates, classified as Ad12 by neutralization tests, were much more related to Ad31 than to Ad12 prototype Huie strain. These DNA homology groupings are consistent in the main with the properties of other “groupings” of human Ads, e.g., oncogenic groups (tumorigenicity in newborn hamsters), T-antigen groups, G + C content of viral DNA, hemagglutination groups, molecular characteristics of subviral particles and virion proteins (e.g., length of fiber), and human epidemiology and pathogenicity.

Biochemical studies on adenovirus multiplication: II. Kinetics of nucleic acid and protein synthesis in suspension cultures☆

Abstract KB cells growing exponentially in suspension were infected with type 2 adenovirus at high input multiplicities. An increase in intracellular virus was first detected at 14 hours after infection and reached levels of 4000–10,000 PFU per cell at 36–48 hours after infection. Uninfected cells multiplied 3- to 4-fold during 48-hour experimental periods. In contrast, multiplication of cells in infected cultures was limited to the first 12 hours and was never more than 1.2- to 1.3-fold. Measurements of virus adsorption, infective center formation, and surviving cells indicated that essentially all cells were infected within 1 hour after addition of virus. Infected cells did not lyse but appeared to increase in size. Dry weight analysis revealed a continuous increase in mass per infected cell to double that of control cells at 36 hours after infection. Protein, DNA, and RNA accumulated continuously in infected cells starting within 12 hours after infection. At 36 hours after infection the content per cell of these polymers reached double that of uninfected cells. The significance of increased polymer synthesis in relation to virus production is discussed.

RNA-Directed DNA Polymerase—Properties and Functions in Oncogenic RNA Viruses and Cells1

Publisher Summary This chapter provides description of the properties and classification of the oncornaviruses and related virus particles that possess RNA→DNA polymerase molecules. The chapter describes the properties of the DNA polymerase activities of oncornaviruses and related particles including the endogenous reaction, the reaction products, and the utilization of external templates. The chapter also deals with the studies on the inhibitors of the viral DNA polymerase, their mechanism of action and their effect on cell transformation and tumor induction. The properties of purified RNA→DNA polymerase and the mechanism of DNA synthesis are also explored. The chapter provides evidence for the in vivo function of the viral RNA→DNA polymerase and a concise description of the present understanding of the molecular events of oncornavirus replication and cell transformation. The chapter also discusses the analysis of viral related base sequences in normal and cancer cells by molecular hybridization with the viral DNA product of the RNA→DNA polymerase, and studies on RNA→DNA polymerase in normal and cancer cells.

Viral RNA Subunits in Cells Transformed by RNA Tumor Viruses

Single-stranded 35S and 20S viral RNA species are synthesized in virus-producing mouse and rat cells transformed by the murine sarcoma virus. A transformed hamster cell line that does not produce virus synthesizes 35S, but not 20S viral RNA.