Paul M. Loewenstein

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.

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.

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.

An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis

We have shown previously that a synthetic peptide of 49 amino acids, encoding mainly adenovirus E1A protein domain 3 (PD3), functions as an autonomous transcriptional activator. Here we provide two lines of evidence showing that E1A transactivation does not require the induction of cellular protein synthesis. First, PD3 rapidly transactivates E1A-inducible early viral genes in the presence of inhibitors of protein synthesis, as demonstrated by microinjection-in situ hybridization experiments. Second, PD3 greatly stimulates transcription of E1A-inducible genes in vitro. Mutant PD3 peptides with single amino acid substitutions in conserved cysteine residues are defective in transactivation both in vivo and in vitro. Our findings provide compelling evidence that protein synthesis is not required for E1A transactivation, and support a model in which E1A modifies the activity of a preexisting cellular protein(s) involved in the regulation of transcription.

The Adenovirus E1A Repression Domain Disrupts the Interaction between the TATA Binding Protein and the TATA Box in a Manner Reversible by TFIIB

The human adenovirus E1A 243 amino acid oncoprotein possesses a transcription repression function that appears to be linked with its ability to induce cell cycle progression and to inhibit cell differentiation. The molecular mechanism of E1A repression has been poorly understood. Recently, we reported that the TATA binding protein (TBP) is a cellular target of E1A repression. Here we demonstrate that the interaction between TBP and the E1A repression domain is direct and specific. The TBP binding domain within E1A 243R maps to E1A N-terminal residues approximately 1 to 35 and is distinct from the TBP binding domain within conserved region 3 unique to the E1A 289R transactivator. An E1A protein fragment consisting of only the E1A N-terminal 80 amino acids (E1A 1-80) and containing the E1A repression function was found to block the interaction between TBP and the TATA box element as shown by gel mobility and DNase protection analysis. Interestingly, a preformed TBP-TATA box promoter complex can be dissociated by E1A 1-80. Further, TFIIB can prevent E1A disruption of TBP-TATA box interaction. TFIIB, like TBP, can overcome E1A repression of transcription in vitro. The ability of the E1A repression domain to block TBP interaction with the TATA box and the ability of TFIIB to reverse E1A disruption of the TBP-TATA box complex implies a mechanism for E1A repression distinct from those of known cellular repressors that target TBP.

Demonstration that a chemically synthesized BPV1 oncoprotein and its C-terminal domain function to induce cellular DNA synthesis.

Bovine papillomavirus type 1 contains the smallest known oncogene (ORF E5), encoding a hydrophobic 44 amino acid protein. To study the biochemical functions of the E5 oncoprotein, we have chemically synthesized it and several deletion mutant peptides. We demonstrate induction of cellular DNA synthesis in growth-arrested cells by microinjection of E5 oncoprotein. This activity can be broken down into two functionally distinguishable domains. Remarkably, the first domain, which alone is sufficient to induce cellular DNA synthesis, contains only the C-terminal 13 amino acids. This is the smallest known protein fragment that can autonomously activate cellular DNA synthesis. The second domain is the hydrophobic middle region, which by itself fails to induce cellular DNA synthesis but confers a 1000-fold increase in specific activity. The N-terminal one-third of the molecule is dispensable for induction of DNA synthesis.

Adenovirus E1A N-Terminal Amino Acid Sequence Requirements for Repression of Transcription In Vitro and In Vivo Correlate with Those Required for E1A Interference with TBP-TATA Complex Formation

ABSTRACT The adenovirus (Ad) E1A 243R oncoprotein encodes an N-terminal transcription repression domain that is essential for early viral functions, cell immortalization, and cell transformation. The transcription repression function requires sequences within amino acids 1 to 30 and 48 to 60. To elucidate the roles of the TATA-binding protein (TBP), p300, and the CREB-binding protein (CBP) in the mechanism(s) of E1A repression, we have constructed 29 amino acid substitution mutants and 5 deletion mutants spanning the first 30 amino acids within the E1A 1-80 polypeptide backbone. These mutant E1A polypeptides were characterized with regard to six parameters: the ability to repress transcription in vitro and in vivo, to disrupt TBP-TATA box interaction, and to bind TBP, p300, and CBP. Two regions within E1A residues 1 to 30, amino acids 2 to 6 and amino acid 20, are critical for E1A transcription repression in vitro and in vivo and for the ability to interfere with TBP-TATA interaction. Replacement of 6Cys with Ala in the first region yields the most defective mutant. Replacement of 20Leu with Ala, but not substitutions in flanking residues, yields a substantially defective phenotype. Protein binding assays demonstrate that replacement of 6Cys with Ala yields a mutant completely defective in interaction with TBP, p300, and CBP. Our findings are consistent with a model in which the E1A repression function involves interaction of E1A with p300/CBP and interference with the formation of a TBP-TATA box complex.

Transcriptional Repression by Human Adenovirus E1A N Terminus/Conserved Domain 1 Polypeptides in Vivo and in Vitro in the Absence of Protein Synthesis

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.

Mapping of HIV-1 tat protein sequences required for binding to tar RNA

We have utilized a gel retardation assay to study the binding of chemically synthesized domains of the HIV-1 Tat protein to radiolabeled trans-activating response element (Tar) RNA. As with recombinant Tat protein, synthetic Tat specifically binds to Tar RNA and not to a defective Tar RNA or to anti-sense Tar RNA. The 6 amino acid portion of the basic region containing five arginines is sufficient to confer Tar binding to overlapping Tat protein fragments; Tat fragments that lack the basic region do not bind Tar. In addition, the basic region alone can also bind Tar RNA; however, binding of the basic region is non specific since defective Tar RNA is bound as well as wild type Tar RNA. Binding specificity for wild type Tar RNA can be conferred by the addition of a minimum of 8 random amino acids to either end of the basic region.

Human Adenoviruses: Propagation, Purification, Quantification, and Storage

Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.