Coxsackievirus B RNA replication: lessons from poliovirus.

The replication of coxsackievirus RNA occurs with rapid onset, starting approximately 2.5 h after infection. The mechanisms entailing the RNA replication of enteroviruses, like coxsackievirus and poliovirus, are highly conserved. These processes require two steps of RNA amplification: (i) complete synthesis of the negative-strand RNA using input RNA as the template and (ii) synthesis of the positive-strand RNA using the intermediate negative-strand RNA as the template. Successful enterovirus RNA replication requires all of the viral nonstructural proteins in their mature and precursor forms, as well as RNA secondary structures in the template. The encoded nonstructural proteins are responsible for RNA replication through multiple protein-protein interactions between viral and/or host proteins to mediate RNA synthesis, induce membranous vesicles, and deliver the replication complex to the template. The RNA secondary structures at the 5' and 3' termini of the template position the RNA replication complex at the initiation site(s) for both negative- and positive-strand RNA synthesis, thus providing binding sites for viral and host proteins that may functionally circularize the genome during RNA synthesis. Although considerable knowledge has been gained regarding the mechanism of enterovirus RNA synthesis, the complete steps in RNA replication have not been fully determined. The aim of this review is to summarize the current state of our knowledge and to present a model that encompasses the identified steps of enterovirus RNA replication.

Insomnia related to dysthymia: polysomnographic and psychometric comparison with normal controls and acute therapeutic trials with trazodone.

Utilizing polysomnography (PSG) and psychometry, objective and subjective sleep and awakening quality was investigated in 11 patients (mean age 50 +/- 14) with nonorganic insomnia (F 51.0) related to dysthymia (F 34.1) as compared with 11 age- and sex-matched normal controls. Patients demonstrated decreased sleep efficiency and sleep stage S2 as well as increased sleep latency to S1, S2 and S3, wakefulness within the total sleep period, number of awakenings, S1 and REM sleep. There was no intergroup difference in REM latency. Subjective sleep quality and the total score of the Self-Assessment Scale for Sleep and Awakening Quality (SSA) were deteriorated as were evening and morning well-being, mood, affectivity and drowsiness. Noopsychic measures showed deteriorated numerical memory, fine motor activity and reaction time variability. In a placebo-controlled crossover design study, the acute effects of 100 mg trazodone, a serotonin reuptake inhibitor with a sedative action due to 5HT(2) and alpha(1) receptor blockade, were investigated in the patients. As compared with placebo, trazodone induced an increase in slow-wave sleep (S3 + 4), a lengthening of REM latency, a decrease in REM sleep and a normalization of the periodic leg movement (PLM) index. In the morning, there was a minimal increase in somatic complaints and a decrease in critical flicker frequency and systolic blood pressure. In conclusion, our study demonstrated that dysthymia induced significant changes in objective and subjective sleep and awakening quality, which were counteracted by 100 mg trazodone, thus suggesting a key-lock principle in the treatment of nonorganic insomnia related to dysthymia with this drug.

[Diagnosis and differential diagnosis of consciousness disorders from the psychiatric viewpoint]. / Diagnose und Differentialdiagnose der Bewusstseinsstörungen aus psychiatrischer Sicht.

Diagnosis and differential diagnosis of disturbance of consciousness are often difficult, especially when occurring in the course of psychiatric diseases. Apart from discord in defining the term of consciousness it is not always easy to distinguish between qualitative and quantitative alterations of the disorders. The performance of an exact interview and a thorough clinical examination allows an estimation of the degree of dimming of consciousness as well as the possible causes. Disorder of Consciousness is often found in patients carrying out substance abuse but also in the course of other psychiatric diseases such as depression, schizophrenia, personality disorders and dissociative disorders. This article is dealing with the clinical approach towards patients experiencing alterations or disturbances of consciousness and is also trying to communicate innovations in the field of psychiatric diagnostics.

[Possible interaction between ethanol and drugs and their significance for drug therapy in the elderly]. / Mögliche Interaktionen zwischen Ethanol und Pharmaka und deren Bedeutung für die medikamentöse Therapie im Alter.

Even though alcohol dependence is not often found in the elderly, alcohol consumption and alcohol abuse are both common. As the elderly also often take medication on a regular basis, this group is at particularly high risk for problems resulting from the concurrent use of these substances. Physical changes as a result of the aging process (e.g. reduction of body water, decrease of hepatic blood flow) and alcohol related diseases can influence the pharmacokinetics and pharmacodynamics of both ethanol as well as other drugs. Alcohol dehydrogenase (ADH), acetaldehydede hydrogenase (ALDH) and cytochrome P450 2E1 are the enzymes responsible for the metabolism of ethanol. These enzymes are also the sites of direct pharmacological interaction between ethanol and other drugs, however, altered effects of medication can also be caused by ethanol adding to or reducing the drug's effect. Although some of these effects result from heavy use of alcohol, others can also occur with moderate use. Interactions have most frequently been described for analgetics, psychopharmacologically active drugs, antihistamines, anticoagulants antihypertensive drugs and antibiotics.

[Management of hallucinating patients]. / Der Umgang mit halluzinierenden Patienten.

Hallucinations are perceptions lacking any extern or physical stimulus. They can affect all senses and may occur in the course of different physical or psychiatric diseases. Patients experiencing hallucinations may derive the conviction that the contents of their hallucinations and the consequent interpretations are real, therefore they may try to convince their relatives and friends. Thus it has proved helpful to follow certain rules in the treatment of these patients. It is important not only to diagnose the basic disorder or illness but also to deal with the patient's anxiety, respect his/her perceptions and to try to establish alternative models of explanation. Apart from therapy of the underlying disorder it is wise to recommend psychotherapy and the attendance of self-help groups.

[Society-research-diagnosis: have classification problems in psychiatry been overcome at the beginning of the new millennium?]. / Gesellschaft--Forschung--Diagnosen: Sind Klassifikationsprobleme in der Psychiatrie nach der Jahrtausendwende überwunden?

Although diagnostics of organic psychoses already had been quite well established at the beginning of the last century, no satisfactory results had been achieved in the field of functional psychoses. This led to a return towards the concept of "unitarian psychoses" and thus to a revival of cross-sectional diagnoses. With the development of psychopharmacological compounds the need for exact demarcation of disease grew. Around this time syndromatological classification systems that were supposed to be used internationally have been developed. In the course of the last thirty years the argument about the meaning of psychiatric diagnoses rose anew; the necessity of standardized means of psychiatric diagnoses was contrasted by the fact that there was no way of defining disease entities for clinical research. A polydiagnostic approach as well as clinical diagnose of the illness course--using single symptoms as predictors of the disease's course--could point to a solution.

Memory deficits in patients with DSM-IV obsessive-compulsive disorder.

Neuropsychological testing provides increasing evidence that certain memory deficits might play an essential role in the emergence of doubts and, as a result, in perpetuating checkers' rituals. Another account of doubting implicates meta-cognitive factors, such as confidence in memory. The present study examined mnestic functioning and self-perception of memory ability in a group of 27 nondepressed patients with obsessive-compulsive disorder (OCD) and 27 normal controls. All patients met DSM-IV and ICD-10 criteria for OCD, displayed prominent behavioral checking rituals and had to show a score on the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) of at least 16. Significant deficits in intermediate (Lern- und Gedächtnistest; LGT-3) and immediate (Corsi Block-Tapping Test) nonverbal memory were identified in the patients with OCD compared to normal controls. Contrary to predictions, OCD patients also showed a significant deficit in general memory and verbal memory (LGT-3). With respect to meta-cognition, OCD patients reported less confidence in their memories than controls. These findings suggest that obsessional doubt reflects a deficit in memory as well as a deficit in memory confidence. Depending on which dysfunction predominates, different therapeutic procedures seem to be required.

Restless legs syndrome (RLS) and periodic limb movement disorder (PLMD): acute placebo-controlled sleep laboratory studies with clonazepam.

Restless legs syndrome (RLS) - a common sensorimotor disorder - and periodic limb movement disorder (PLMD) are currently treated with substances of four classes: dopaminergic agents, which are considered the drugs of choice, benzodiazepines, opioids and anticonvulsants. As their effects on sleep variables differ considerably, the aim of the present placebo-controlled sleep laboratory study was to measure the acute effects of 1 mg clonazepam on objective and subjective sleep and awakening quality in ten RLS and 16 PLMD patients, utilizing polysomnography (PSG) and psychometry. Descriptive data analysis demonstrated at the confirmatory level concerning three target variables that - as compared with placebo - clonazepam significantly improved objective sleep efficiency and subjective sleep quality in both patient groups, but failed to reduce the index PLM/h of sleep. At the descriptive level, in PLMD clonazepam improved PLM during time in bed, REM and wakefulness and showed more significant changes in various sleep and awakening measures than in RLS patients, though there were no significant inter-group differences. In conclusion, in both PLMD and RLS clonazepam exhibited acute therapeutic efficacy regarding insomnia, which is quite different from the mode of action of dopamine agonists.

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.

The European acamprosate trials: conclusions for research and therapy.

In an excellent methodological approach, the European acamprosate study project showed that acamprosate increases sobriety times. In one randomized prospective study (n = 260) comparing acamprosate and placebo, with a 1-year treatment phase and 1-year follow-up phase, the authors found that acamprosate is effective only in Lesch type I and type II patients. To investigate the possible influence of diagnostic subgrouping, we applied the Lesch typology in a co-work with the main researchers of the UK study. The UK results concerning acamprosate's effects in the types do not mirror the Vienna results, but the numbers of type I and type II patients, retrospectively found as included in the UK centers, were too small for any conclusions. The distribution of the types points to the fact that too many type III and IV patients had been included to give acamprosate the chance to be effective. Following our typology and also these studies, we developed special treatment approaches. For relapse prevention studies, the cumulative abstinence duration together with the Lesch typology seems to be promising.

Dopamine and alcohol relapse: D1 and D2 antagonists increase relapse rates in animal studies and in clinical trials.

A considerable number of animal studies on the effects of dopaminergic agents on alcohol intake behavior have been performed. Acute alcohol administration in rats induces dopamine release in the caudate nucleus and in the nucleus accumbens, an effect related among others to reinforcement. It has been repeatedly suggested that D1 and D2 receptor activation mediates reward. As alcohol consumption and dopaminergic transmission seem to have a close relationship, all kinds of dopaminergic agents may be regarded as putative therapeutics for preventing relapse. In a prospective European double-blind multicenter clinical trial, comparing the D1, D2, D3 antagonist flupenthixol and placebo in 281 chronic alcohol-dependent patients (27.4% women), the application of the Lesch typology made an outcome differentiation possible. It could be shown in which patients flupenthixol administration was followed by a significantly higher relapse rate and in which patient groups no differences were found when compared to placebo.

Sleep laboratory studies in periodic limb movement disorder (PLMD) patients as compared with normals and acute effects of ropinirole.

Periodic limb movement disorder (PLMD) occurs in a variety of sleep disorders and can cause insomnia as well as hypersomnia with daytime somnolence. The aim of this study was to investigate 12 untreated PLMD patients as compared with 12 normal controls and to measure the acute effects of 0.5 mg ropinirole (Requip((R))) - a non-ergoline dopamine agonist - as compared with placebo. In three nights (adaptation, placebo, ropinirole night) objective and subjective sleep and awakening quality were evaluated. In the target variable 'periodic leg movements per hour of sleep' (PLM/(hTST)) PLMD patients showed an increased value of 42/h (normal 0-5/h) with a greater number of arousals due to periodic leg movements (PLM) in sleep. They further demonstrated an increased number of awakenings, sleep stage S1, S4, stage shifts and decreased S2, but there were no significant differences concerning total sleep time, sleep efficiency (SE), subjective sleep quality and morning measures of mood, drive and drowsiness. However, measures of attention variability, numerical memory, fine motor activity and reaction time performance were impaired. Ropinirole 0.5 mg was shown to significantly improve the index PLM/(hTST) by 64% and arousals due to PLM, increase spontaneous arousals, REM-latency, stage 2 and stage shifts and decrease SREM. In the morning attention variability was attenuated and numerical memory augmented. Thus, ropinirole improved some sleep architecture and early morning measures of performance but specifically all PLM variables, which suggests a dopaminergic pathogenesis in PLMD. Copyright 2001 John Wiley & Sons, Ltd.

Sensitivity and specificity of carbohydrate-deficient transferrin in drinking experiments and different patients.

Information provided by patients about the amounts of alcohol they drink may often be too subjective and therefore unreliable. Because of the possible serious consequences of interactions between alcohol and medication, reliable laboratory test markers for alcohol consumption are needed. Carbohydrate-deficient transferrin (CDT) is at present the best available objective measure of drinking behavior. During a withdrawal trial, 92 alcohol-dependent patients who had been admitted to a hospital in an ethanol-intoxicated state were monitored over the following 28 days by using the percent carbohydrate-deficient transferrin (%CDT of total transferrin) (%CDT) method. At the time of admission, 63% showed elevated %CDT levels. After a subsequent period of abstinence, a decrease in %CDT levels was apparent in four different groups of patients, whereas in two groups, comprising the greatest number of patients, normal %CDT levels were evident after 14 days of abstinence. In patients whose CDT levels were very high at study initiation, it took at least 21 to 28 days--and sometimes longer--for CDT to decrease to the radioimmunoassay (RIA) %CDT test cutoff point of 2.5. In a further study of 56 male alcohol-dependent patients, we measured liver enzyme concentrations, mean corpuscular volume (MCV), and four CDT variants on the first day of evidence of withdrawal syndrome. We found a significant correlation between results on the Munich Alcoholism Test (MALT) and MCV levels; among gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels; and among all four CDT variants. A cluster analysis yielded three clusters: (1) GGT, AST, and ALT levels; (2) MCV levels and MALT results; and (3) all CDT measurement variants. We conclude that these three clusters measure different detriments to the patient and that all available CDT variants are commensurate.

A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice.

The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5' NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements.

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 5' noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.

Pyrimidine-rich region mutations compensate for a stem-loop V lesion in the 5' noncoding region of poliovirus genomic RNA.

Five revertants of a linker-scanning mutation adjacent to the stem-loop V attenuation determinant (X472) in the 5' noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs with seven nucleotide changes in the pyrimidine-rich region. Generation of the identical rare second-site mutations suggests the existence of a replicase-dependent mutagenesis mechanism during poliovirus replication. Enzymatic structure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary structure model is presented for wild-type stem-loops V and VI and the pyrimidine-rich region located in the 5' noncoding region of poliovirus RNA. A pyrimidine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences were not due to cell-type differences in viral translation or RNA replication. Release of X472-R4N from HeLa cells was 10-fold lower than release from neuroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently from neuroblastoma cells (approximately 4-fold increase compared with release from HeLa cells), indicating that poliovirus neurotropism may be influenced by the cell-type efficiency of virus release. Thermal treatment increased the levels of infectious X472-R4N virions but not wild-type virus particles; thus RNA sequence and structural changes in the mutated 5' noncoding region of X472-R4N may have altered RNA-protein interactions necessary for virus infectivity.

Requirements for RNA replication of a poliovirus replicon by coxsackievirus B3 RNA polymerase.

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D(pol)) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D(pol) from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5' end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33 degrees C, but not at 37 degrees C. Replacement of the PV1 5'-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.

Modulation of the RNA binding and protein processing activities of poliovirus polypeptide 3CD by the viral RNA polymerase domain.

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5' RNA sequences.

RNA structure adjacent to the attenuation determinant in the 5'-non-coding region influences poliovirus viability.

In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.

Translation initiation of a cardiac voltage-gated potassium channel by internal ribosome entry.

The mammalian Kv1.4 voltage-gated potassium channel mRNA contains an unusually long (1.2 kilobases) 5'-untranslated region (UTR) and includes 18 AUG codons upstream of the authentic site of translation initiation. Computer-predicted secondary structures of this region reveal complex stem-loop structures that would serve as barriers to 5' --> 3' ribosomal scanning. These features suggested that translation initiation in Kv1.4 might occur by the mechanism of internal ribosome entry, a mode of initiation employed by a variety of RNA viruses but only a limited number of vertebrate genes. To test this possibility we introduced the 5'-UTR of mouse Kv1.4 mRNA into the intercistronic region of a bicistronic vector containing two tandem reporter genes, chloramphenicol acetyltransferase and luciferase. The control construct translated only the upstream chloramphenicol cistron in transiently transfected mammalian cells. In contrast, the construct containing the mKv1.4 UTR efficiently translated the luciferase cistron as well, demonstrating the presence of an internal ribosome entry segment. Progressive 5' --> 3' deletions localized the activity to a 3'-proximal 200-nucleotide fragment. Suppression of cap-dependent translation by extracts from poliovirus-infected HeLa cells in an in vitro translation assay eliminated translation of the upstream cistron while allowing translation of the downstream cistron. Our results indicate that the 5'-untranslated region of mKv1.4 contains a functional internal ribosome entry segment that may contribute to unusual and physiologically important modes of translation regulation for this and other potassium channel genes.