Biomedical subjects

V G Wilson

Publications and source records attributed to V G Wilson.

At least 19 recordsLinked to original sources

PubMed2005

Comparison of the effects of nitric oxide donors and calcium channel blockers on the intrinsic myogenic tone of sheep isolated internal anal sphincter.

BACKGROUND: Chronic anal fissure is associated with considerable pain and anal hypertonia. Numerous clinical studies attest to the effectiveness of individual nitro-containing drugs and organic calcium channel blockers in this condition but there are few comparative studies. METHODS: Isolated segments of sheep internal anal sphincter were prepared for isometric tension recording. The effect of various drugs on myogenic tone was examined in the absence or presence of sodium orthovanadate (SOV), an agent used to mimic anal hypertonia by increasing myogenic tone. RESULTS: All the drugs tested produced concentration-dependent inhibition of myogenic tone, with the maximum effect ranging from 66.4 per cent (verapamil) to 100 per cent (sodium nitroprusside). Sodium nitroprusside and diltiazem were the most potent, followed by glyceryl trinitrate (GTN), nifedipine and verapamil, which had similar potency, and finally nicorandil. The potency of GTN and diltiazem was reduced threefold in the presence of 1 mmol/l SOV. The combined effect of GTN and diltiazem was greater than the effect of either agent alone, even in the presence of 3 mmol/l SOV. CONCLUSION: Nitro-containing drugs and organic calcium channel blockers are potent inhibitors of anal sphincter myogenic tone that may be used in combination to treat chronic anal fissure.

Anal Canal↗

PubMed2003

L-arginine-induced relaxation of the internal anal sphincter is not mediated by nitric oxide.

BACKGROUND: Topical application of L-arginine, the precursor of nitric oxide, reduces resting anal pressure without significant side-effects and may therefore be of benefit in the treatment of anal fissure. This in vitro study investigated the effect of L-arginine on sheep and human isolated internal anal sphincter (IAS) to ascertain the role played by nitric oxide and guanosine 3',5'-cyclic monophosphate. METHODS: Strips of sheep and human IAS were mounted in isolated organ baths. The effects on myogenic tone of increasing concentrations of L-arginine, D-arginine and other amino acids were evaluated. RESULTS: L-Arginine, D-arginine and other basic amino acids (L-lysine and L-ornithine) all caused a concentration-dependent reduction in myogenic tone. L-Arginine was the most effective and produced a mean(s.e.m.) maximal reduction in myogenic tone of 78.2(7.1) and 40.2(9.3) per cent in sheep and human tissue respectively. These responses were not affected by N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase. Changes in pH per se were unable to explain the relaxation fully, but an equiosmolar sodium chloride solution produced a concentration-response relationship similar to that of L-arginine. CONCLUSION: The ability of L-arginine to reduce myogenic tone is independent of nitric oxide. This effect may be partially pH dependent but the osmolality of the solution appears to be a major factor. Hyperosmolar solutions might be worthy of further investigation as agents that affect anal tone.

Anal Canal↗

PubMed2003

No evidence for activation of alpha(2)-adrenoceptors by methanolic extracts of bovine brain and lung containing clonidine-displacing substance.

Methanolic extracts of bovine brain and lung are capable of displacing [(3)H]-clonidine from alpha(2)-adrenoceptor binding sites, indicating the presence of a clonidine-displacing substance (CDS). We have examined alpha(2)-adrenergic responses and the extracts in three models: [(3)H]-cyclic AMP accumulation in miniprisms of guinea pig cerebral cortex, isometric tension measurements of isolated segments of the rat vas deferens, and porcine palmar lateral vein. The selective alpha(2)-adrenoceptor agonist, 5-bromo-6-2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304) inhibited forskolin-stimulated [(3)H]-cyclic AMP accumulation in the cerebral cortex and elicited contractions of the porcine isolated palmar lateral vein. Clonidine (0.1-30 nM) inhibited neurogenic contractions of the rat vas deferens. Responses to both agonists were inhibited by the alpha(2)-adrenoceptor antagonists, idazoxan or rauwolscine. Brain CDS (5 units/mL) reduced forskolin-stimulated [(3)H]-cyclic AMP accumulation in the guinea pig cerebral cortex, whereas lung CDS (1 unit/mL) increased the accumulation of the cyclic nucleotide. Neither response to the extracts was inhibited by 1 microM idazoxan. Low concentrations of both extracts (0.05 unit/mL) reduced electrically evoked contractions of the rat vas deferens by approximately 20%, but higher concentrations enhanced neurogenic contractions by approximately 50%. Again, the effect of the brain extract was not altered by 1 microM idazoxan. Lung CDS (0.02-1 unit/mL) induced contractions of the porcine palmar lateral vein that were also insensitive to rauwolscine. The results suggest that brain and lung CDS do not activate either central or peripheral alpha(2)-adrenoceptors.

Adrenergic alpha-Agonists↗

PubMed2003

Complex interaction of alpha(2)-adrenoceptor binding sites with bovine brain and lung extracts containing clonidine-displacing substance.

Previous studies have established that methanolic extracts of bovine brain and lung possess CDS activity with an agonist-like profile at alpha(2)-adrenoceptor binding sites (i.e., greater potency against [(3)H]-clonidine sites than against [(3)H]-RX-821001 sites). Following prolonged reflux in ethylacetate, monovalent cations and histamine were removed from the lung extract, and the resulting extract exhibited similar potency against both radioligands, indicative of an antagonist-like profile. These observations help explain the absence of biologic activity at alpha(2)-adrenoceptors in the methanolic extracts as reported in our companion paper.

Animals↗

PubMed2003

Evidence for nonadrenoceptor responses to imidazoline derivatives in the porcine isolated rectal artery.

High concentrations of phentolamine, efaroxan, and idazoxan were found to produce nonadrenoceptor contractions of the porcine isolated rectal artery previously exposed to U46619 and forskolin. These responses were insensitive to the putative imidazoline I(3) receptor antagonist KU-14R, unlike those previously reported in this preparation for oxymetazoline. The pharmacologic nature of this response and the obligate requirement for preconstriction suggests that these imidazoline derivatives modulate ion channel function through a novel nonadrenergic site.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2003

Molecular targets for papillomavirus therapy.

Papillomaviruses are infectious agents for human and animal epithelial tissue, and nearly 100 distinct human types (HPVs) have been identified. When these viruses infect cutaneous or mucosal skin they can initially cause clinical warts or persistent infection with little or no visible manifestations. Warts, while usually benign, can be painful or cosmetically unacceptable and often require medical treatment. Furthermore, infection with certain specific HPV types, such as 16 or 18 (as well as several others), is the major risk factor for a woman's development of cervical cancer. In addition to cervical cancer, papillomaviruses have also been implicated in cancers of the skin and respiratory track though the evidence is not yet as conclusive. It is clear that prevention or elimination of papillomavirus infections would ultimately reduce the incidence of cervical cancer and possibly other epithelial cancers as well. Unfortunately, progress in vaccine development has been slow and no specific anti-papillomavirus agents are available. The rational development of effective anti-papillomaviral treatments will require a detailed understanding of how these viruses replicate and interact with the host cell, and much progress has been made in this area over the last 10 years. These viruses have small DNA genomes with limited coding capacity, and their complete array of viral protein products is known. This review will discuss the known functions of the viral proteins with a focus on strategies to interdict their biological activities as a possible means of specific therapy.

Antiviral Agents↗

PubMed2001

Viral interaction with the host cell sumoylation system.

A novel host cell post-translational modification system termed sumoylation was discovered recently. Sumoylation is an enzymatic process that is biochemically analogous to, but functionally distinct from ubiquitinylation. As in ubiquitinylation, sumoylation involves the attachment of a small protein moiety, SUMO, to substrate proteins. Conjugation of SUMO does not typically lead to degradation of the substrate and instead causes functional alterations or changes in intracellular localization. While the majority of identified SUMO targets are cellular proteins, both herpesvirus and papillomavirus proteins have also been identified as authentic substrates for this modification. The exact effect of sumoylation on viral proteins appears to be substrate specific, but does have functional consequences that are likely to be important for the viral life cycle. In addition to viral proteins being targets for sumoylation, there is both direct and indirect evidence that viruses can alter the sumoylation status of host cell proteins. Such modulation of critical host proteins may be important for inhibiting cellular defense mechanisms or for promoting an intracellular state that is supportive of viral reproduction. This review highlights the enzymology of sumoylation and discusses the known examples of how viruses impact and are impacted by sumoylation.

Animals↗

PubMed2001

Intracellular targeting of proteins by sumoylation.

A novel host cell posttranslational modification system, termed sumoylation, has recently been characterized. Sumoylation is an enzymatic process that is biochemically analogous to, but functionally distinct from, ubiquitinylation. As in ubiquitinylation, sumoylation involves the covalent attachment of a small protein moiety, SUMO, to substrate proteins. However, conjugation of SUMO does not typically lead to degradation of the substrate and instead has a more diverse array of effects on substrate function. As the list of sumoylation substrates has expanded, a common theme is that many substrates exhibit sumoylation-dependent subcellular distribution. While the molecular mechanisms by which sumoylation targets protein localization are still poorly understood, it is clear that this modification system is an important regulator of intracellular protein localization, particularly involving nuclear uptake and punctate intranuclear accumulation.

Drosophila Proteins↗

PubMed2001

Evidence for a non-adrenoceptor, imidazoline-mediated contractile response to oxymetazoline in the porcine isolated rectal artery.

Imidazoline derivatives are known to elicit responses through both alpha(2)-adrenoceptor and non-adrenoceptor, imidazoline sites, though as yet there are no examples of the latter on vascular smooth muscle. In the presence of 0.3 microM prazosin, neither UK-14304 (0.01 - 3 microM) nor oxymetazoline (0.01 - 30 microM) caused a significant contraction of the porcine isolated rectal artery, a preparation with a low density of alpha(2)-adrenoceptors. In the presence of a combination of U46619 and forskolin, however, both agonists produced concentration-dependent contractions. Pretreatment with phenoxybenzamine (3 microM) abolished responses to UK-14304, but left those elicited by oxymetazoline largely unaffected. The putative I(3) imidazoline antagonist 2-(2,3 dihydro-2-benzofuranyl)-2-imidazole (KU-14R, 10 microM) caused a 6 fold rightward displacement of the phenoxybenzamine-insensitive concentration - response curve to oxymetazoline. Our data indicates that non-adrenoceptor, imidazoline sites, pharmacologically similar to the I(3) imidazoline site on islet cells, mediate vasoconstriction in the porcine isolated rectal artery.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2001

Detection and heterogeneity of herpesviruses causing Pacheco's disease in parrots.

Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses.

Animals↗

PubMed2001

Functional mapping of the DNA binding domain of bovine papillomavirus E1 protein.

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).

Amino Acid Sequence↗

PubMed2000

SUMO-1 modification of bovine papillomavirus E1 protein is required for intranuclear accumulation.

The E1 protein is a multifunctional, origin-binding helicase that is essential for replication of papillomaviruses. Recently, bovine papillomavirus E1 was shown to be post-translationally modified by the addition of the SUMO-1 polypeptide. Here we show that the site of sumoylation maps to lysine residue 514. This lysine and the flanking sequences are well conserved in human papillomavirus (HPV) E1 proteins. Both HPV1a and HPV18 E1 proteins are substrates for sumoylation in vitro, which is consistent with this modification being a general property of E1 proteins. Mutations, which impair the sumoylation of bovine papillomavirus E1, prevent normal nuclear accumulation of E1 with a concomitant loss of replication capacity. These results suggest that sumoylation plays a role in nuclear transport and could regulate the E1 replication function by controlling access to the nuclear replication domains.

Animals↗

PubMed2000

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein.

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.

Amino Acid Sequence↗

PubMed2000

Pharmacological studies on the inhibitory action of melatonin and putative melatonin analogues on porcine vascular smooth muscle.

The effect of high concentrations of melatonin, and related indole-based and naphthalene-based derivatives, has been examined in the porcine coronary artery, pulmonary artery and the marginal artery of the colon. In addition, we have pharmacologically examined the role of cyclic GMP in the relaxatory action of these agents. Cumulative addition of melatonin (3-300 microM) caused a slowly developing relaxation in all three vascular preparations pre-contracted with 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2alpha (U46619), a thromboxane mimetic agent. The estimated pIC50 values were 4.10-3.70 (coronary artery), 3.89 (pulmonary artery) and 3.96 (marginal artery). All melatonin analogues examined also produced concentration-dependent inhibition of U46619-induced contractions of the coronary and marginal arteries in a qualitatively similar manner to melatonin. The rank order of potency (based on the pIC50 values) of these compounds in both vascular tissues was N-[2-(3-ethyl-7-methoxynaphthyl) ethyl]-acetamide (S21634) >2-iodomelatonin = N-[2-(7-methoxynaphth-1-yl)-ethyl]-acetamide (S20098) = N-[2-naphth-1-yl-ethyl]-cyclobutyl carboxamide (S20928) >melatonin >N-acetyl-5-HT. Finally, the pharmacological characteristics of melatonin and S21634 as phosphodiesterase inhibitors were compared to those of zaprinast, a known cyclic GMP-specific phosphodiesterase inhibitor. Zaprinast also caused concentration-dependent inhibition of U46619-induced tone. All three compounds, zaprinast (10 microM), melatonin (300 microM) and S21634 (30 microM), significantly enhanced sodium nitroprusside-induced relaxations. The inhibitory action of zaprinast per se was greater in the presence of the endothelium and significantly attenuated by 3 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective inhibitor of guanylyl cyclase. In marked contrast, the vasorelaxant action of melatonin and S21634 was not affected by the removal of the endothelium or the addition of ODQ. In summary, we have shown that porcine arterial smooth muscle relaxes in response to high concentrations of melatonin and other related melatonin receptor ligands. However, it appears that the receptive site is pharmacologically different from the melatonin receptors currently known and does not involve inhibition of cyclic GMP-specific phosphodiesterase.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Pharmacological comparison of the effect of ibogaine and 18-methoxycoronaridine on isolated smooth muscle from the rat and guinea-pig.

Ibogaine and 18-methoxycoronaridine are naturally occurring alkaloids reported to possess antiaddictive properties in several models of drug dependence. We have examined their effect at mu-opioid receptors regulating neurogenic contractions of several smooth muscle preparations and also against spontaneous contractions of the rat isolated portal vein. Ibogaine (pIC(50) 5.28) and 18-methoxycoronaridine (pIC(50) 5.05) caused a concentration-dependent inhibition of cholinergic contractions of the guinea-pig ileum which was not affected by the opioid receptor antagonist naloxone (1 microM). In the rat isolated vas deferens ibogaine and 18-methoxycoronaridine caused a concentration-dependent enhancement of purinergic contractions. Both agents (30 microM) caused a 3 - 5 fold rightward displacement of DAMGO-induced inhibition of purinergic contractions, but similar effects were observed for ibogaine against alpha(2)-adrenoceptor-mediated inhibition of neurogenic responses. In the guinea-pig isolated bladder both ibogaine (10 microM) and 18-methoxycoronaridine (10 microM) caused a 2 fold increase in the purinergic component of neurogenic contractions without significantly altering cholinergic contractions or responses to exogenous ATP. In contrast, ibogaine (1 - 30 microM), but not 18-methoxycoronaridine, caused a concentration-dependent enhancement of spontaneous contractions of the rat isolated portal vein. In summary, while ibogaine and 18-methoxycoronaridine modulated electrically-evoked contractions in the three preparations examined, we have no evidence for a selective interaction with pre-junctional mu-opioid receptors. The pronounced enhancement of purinergic contractions produced by both agents is a novel finding and worthy of further investigation.

Animals↗

PubMed2000

Pharmacological characterization of neurogenic responses of the sheep isolated internal anal sphincter.

The aim of the study was to establish the nature of the neurogenic responses of the sheep isolated anal sphincter. Isolated strips of sheep internal anal sphincter develop intrinsic contractile tone following the application of stretch tension. On transmural stimulation (1 - 20 Hz, 10 V pulse strength, 0.5 ms pulse width, 1 s every 180 s) transient relaxations were observed. The amplitude of the relaxations were frequency-dependent reaching a maximal response at 10 - 20 Hz and were inhibited by tetrodotoxin (0.3 microM). Neither atropine (0.3 microM) nor phentolamine (1 microM) affected control responses. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and the selective inhibitor of soluble guanylyl cyclase ODQ, (1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one) (1 microM) completely inhibited the neurogenic relaxations and uncovered contractions that were abolished by 1 microM phentolamine and 0.1 microM prazosin. The effect of L-NAME, but not that of ODQ, was partially reversed by the addition of L-arginine (1 mM). Sodium nitroprusside (10 nM - 10 microM) caused concentration-dependent inhibition of myogenic tone and this effect was significantly reduced by ODQ. Calcium-free Krebs-Henseleit solution also reduced myogenic tone by 85%. Transmural electrical stimulation of the sheep isolated internal anal sphincter causes a transient relaxation of myogenic tone that appears to involve nitric oxide from non-adrenergic, non-cholinergic nerves and, to a lesser degree, noradrenaline from sympathetic nerves. The characteristics of the preparation compares well with that of human tissue and may prove to be a suitable animal based model for further studies.

Anal Canal↗

PubMed2000

Pharmacological examination of contractile responses of the guinea-pig isolated ileum produced by mu-opioid receptor antagonists in the presence of, and following exposure to, morphine.

We have assessed the potential of several mu-opioid receptor antagonists to elicit a response in the guinea-pig isolated ileum in the presence of, and following overnight exposure to, morphine. Naloxone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), (-)-5, 9alpha-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorphan (MR2266), but not D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), produced a transient inhibition of electrically-evoked contractions of the guinea-pig ileum. The effect of 1 microM CTOP, but not that to MR2266, was inhibited by 1 microM somatostatin. Naloxone (0.3 microM), CTOP (3 microM), CTAP (3 microM) and MR2266 (0.3 microM) antagonized the inhibitory effect of morphine on electrically-evoked contractions of the guinea-pig to a similar degree and, following 60 min exposure to morphine, produced non-sustained contractions. The response to 3 microM CTOP was significantly smaller than that to 3 microM CTAP. None of the antagonists produced a response in the absence of morphine. Following overnight exposure of the ileum to 0.3 microM morphine (4 degrees C), and repeated washing to remove the agonist, all four antagonists elicited non-sustained contractions. However, the responses to 3 microM CTOP and 0.3 microM MR2266 were significantly smaller than those elicited by 0.3 microM naloxone and 3 microM CTAP. Somatostatin (1 microM) significantly reduced naloxone-induced contractions, but not those to CTAP. While all four mu-opioid antagonists elicited contractions in the presence of, and following prolonged exposure to, morphine, differences between them were noted which may be a consequence of non-opioid actions.

Animals↗

PubMed2000

Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein.

The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication. Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro. To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features. The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins. E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding. Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected. Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity. To examine E1 binding in vivo, a yeast one-hybrid system was developed. Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction. Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts.

Amino Acid Sequence↗