Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment)-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT) showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3') were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV)) and one retrotransposon (Ty3) RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'). In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3') showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs) mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

The integrity of the periplasmic domain of the VirA sensor kinase is critical for optimal coordination of the virulence signal response in Agrobacterium tumefaciens.

The plant pathogen Agrobacterium tumefaciens responds to three main signals at the plant-bacterium interface: phenolics, such as acetosyringone (AS), monosaccharides, and acidic pH (∼5.5). These signals are transduced via the chromosomally encoded sugar binding protein ChvE and the Ti plasmid-encoded VirA/VirG two-component regulatory system, resulting in the transcriptional activation of the Ti plasmid virulence genes. Here, we present genetic and physical evidence that the periplasmic domain of VirA dimerizes independently of other parts of the protein, and we examine the effects of several engineered mutations in the periplasmic and transmembrane regions of VirA on vir-inducing capacity as indicated by AS sensitivity and maximal level of vir-inducing activity at saturating AS levels. The data indicate that helix-breaking mutations throughout the periplasmic domain of VirA or mutations that reposition the second transmembrane domain (TM2) of VirA relieve the periplasmic domain's repressive effects on the maximal activity of this kinase in response to phenolics, effects normally relieved only when ChvE, sugars, and low pH are also present. Such relief, however, does not sensitize VirA to low concentrations of phenolics, the other major effect of the ChvE-sugar and low pH signals. We further demonstrate that amino acid residues in a small Trg-like motif in the periplasmic domain of VirA are crucial for transmission of the ChvE-sugar signal to the cytoplasmic domain. These experiments provide evidence that small perturbations in the periplasmic domain of VirA can uncouple sugar-mediated changes in AS sensitivity from the sugar-mediated effects on maximal activity.

Molecular basis of ChvE function in sugar binding, sugar utilization, and virulence in Agrobacterium tumefaciens.

ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners.

Novel aptamer inhibitors of human immunodeficiency virus reverse transcriptase.

Primer-template-based double-stranded nucleic acids capable of binding human immunodeficiency virus reverse transcriptase (HIV-RT) with high affinity were used as starting material to develop small single-stranded loop-back DNA aptamers. The original primer-templates were selected using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach and consisted of 46- and 50-nt primer and template strands, respectively. The major determinant of the approximately 10-fold tighter binding in selected sequences relative to control primer-templates was a run of 6.8 G residues at the 3' primer end. Sixty, thirty-seven, twenty-seven, and twenty-two nucleotide loop-back single-stranded versions that retained the base pairs near the 3' primer terminus were constructed. Both the 60- and 37-nt versions retained high affinity for RT with K(d) values of approximately 0.44 nM and 0.66 nM, respectively. Random sequence primer-templates of the same length had K(d)s of approximately 20 nM and approximately 161 nM. The shorter 27- and 22-nt aptamers bound with reduced affinity. Several modifications of the 37-nt aptamer were also tested including changes to the terminal 3' G nucleotide and internal bases in the G run, replacement of specific nucleotides with phosphothioates, and alterations to the 5' overhang. Optimal binding required a 4- to 5-nt overhang, and internal changes within the G run had a pronounced negative effect on binding. Phosphothioate nucleotides or the presence of a 3' dideoxy G residue did not alter affinity. The 37-nt aptamer was a potent inhibitor of HIV-RT in vitro and functioned by blocking binding of other primer-templates.

Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58.

Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence. Earlier studies indicate that the att (attachment) genes of A. tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants. Mutations in many att genes, notably attR and attD, rendered the strain avirulent. These genes are located on pAtC58. Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence. We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence. Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a PvirB::lacZ fusion.