Regulation of the expression and activity of Unr in mammalian cells.

Unr (upstream of N-ras) is a post-transcriptional regulator of gene expression, essential for mammalian development and mutated in many human cancers. The expression of unr is itself regulated at many levels; transcription of unr, which also affects expression of the downstream N-ras gene, is tissue and developmental stage-dependent and is repressed by c-Myc and Max (Myc associated factor X). Alternative splicing gives rise to six transcript variants, which include three different 5'-UTRs. The transcripts are further diversified by the use of three alternative polyadenylation signals, which governs whether AU-rich instability elements are present in the 3'-UTR or not. Translation of at least some unr transcripts can occur by internal initiation and is regulated in a cell-cycle-dependent manner; binding of PTB (polypyrimidine tract-binding protein) and Unr to the 5'-UTR inhibits translation, but these are displaced by heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNPC1/C2) during mitosis to stimulate translation. Finally, Unr is post-translationally modified by phosphorylation and lysine acetylation, although it is not yet known how these modifications affect Unr activity.

Post-transcriptional regulation of gene expression by Unr.

Unr (upstream of N-ras) is a eukaryotic RNA-binding protein that has a number of roles in the post-transcriptional regulation of gene expression. Originally identified as an activator of internal initiation of picornavirus translation, it has since been shown to act as an activator and inhibitor of cellular translation and as a positive and negative regulator of mRNA stability, regulating cellular processes such as mitosis and apoptosis. The different post-transcriptional functions of Unr depend on the identity of its mRNA and protein partners and can vary with cell type and changing cellular conditions. Recent high-throughput analyses of RNA-protein interactions indicate that Unr binds to a large subset of cellular mRNAs, suggesting that Unr may play a wider role in translational responses to cellular signals than previously thought.

Overlapping signals for translational regulation and packaging of influenza A virus segment 2.

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.

Maternal vaccination provision in NHS maternity trusts across England.

BACKGROUND: Vaccinations for seasonal influenza and pertussis have been recommended for pregnant women in England since 2010 and 2012, respectively. Uptake rates are suboptimal with large regional variations. To improve uptake, from 2016 onwards maternity trusts were commissioned to offer pertussis (and other) vaccinations in addition to these being available in primary care. Since 2021, Covid-19 vaccination has also been recommended for pregnant women. Overall maternal vaccination rates are routinely available, but not the relative provision by maternity trusts. We aimed to describe the national picture of maternity trust provision of maternal vaccinations, including how the maternity trust vaccination programme has progressed. METHODS: Cross-sectional survey plus comparisons with 2017-18 figures for maternity trust provision of pertussis vaccination, and with UKHSA data for total pertussis vaccination. RESULTS: Twelve NHS commissioners participated (from 13/06/22 to 31/03/23) providing data for 120 (of a total 124) maternity trusts across England. All 120 (100%) trusts were commissioned to deliver influenza, and 107 (89%) to deliver pertussis vaccinations, though not all actually administered the vaccines; 29% offered Covid-19 vaccinations. For 2021-22 we found a mean of 25% (range 0-81.3%) women were vaccinated for pertussis (a large increase compared with previous estimates for 2017-18); and 11% (range 0-74.2%) for influenza, via their maternity trust. Commissioners reported a negative impact of the pandemic on routine vaccination provision. There was indication of efficiency by vaccinating women attending for other appointments. There are diverse mechanisms for reporting pertussis and influenza vaccinations administered at maternity trusts back to primary care, which may be inefficient for maternity staff workload and accuracy of data transfer (especially for pertussis). CONCLUSION: A high proportion of maternity trusts provide both pertussis and influenza vaccinations, despite a negative impact of the pandemic. Reasons for large between-trust variation in vaccination rates should be explored to improve uptake and equity.

International adaptation and validation of the Pro-VC-Be: measuring the psychosocial determinants of vaccine confidence in healthcare professionals in European countries.

BACKGROUND: Healthcare professionals (HCPs) play an important role in vaccination; those with low confidence in vaccines are less likely to recommend them to their patients and to be vaccinated themselves. The study's purpose was to adapt and validate long- and short-form versions of the International Professionals' Vaccine Confidence and Behaviors (I-Pro-VC-Be) questionnaire to measure psychosocial determinants of HCPs' vaccine confidence and their associations with vaccination behaviors in European countries. RESEARCH DESIGN AND METHODS: After the original French-language Pro-VC-Be was culturally adapted and translated, HCPs involved in vaccination (mainly GPs and pediatricians) across Germany, Finland, France, and Portugal completed a cross-sectional online survey in 2022. A 10-factor multigroup confirmatory factor analysis (MG-CFA) of the long-form (10 factors comprising 34 items) tested for measurement invariance across countries. Modified multiple Poisson regressions tested the criterion validity of both versions. RESULTS: 2,748 HCPs participated. The 10-factor structure fit was acceptable to good everywhere. The final MG-CFA model confirmed strong factorial invariance and showed very good fit. The long- and short-form I-Pro-VC-Be had good criterion validity with vaccination behaviors. CONCLUSION: This study validates the I-Pro-VC-Be among HCPs in four European countries; including long- and short-form tools for use in research and public health.

Prospective Study of the Performance of Parent-Collected Nasal and Saliva Swab Samples, Compared with Nurse-Collected Swab Samples, for the Molecular Detection of Respiratory Microorganisms.

Respiratory tract infections (RTIs) are ubiquitous among children in the community. A prospective observational study was performed to evaluate the diagnostic performance and quality of at-home parent-collected (PC) nasal and saliva swab samples, compared to nurse-collected (NC) swab samples, from children with RTI symptoms. Children with RTI symptoms were swabbed at home on the same day by a parent and a nurse. We compared the performance of PC swab samples as the test with NC swab samples as the reference for the detection of respiratory pathogen gene targets by reverse transcriptase PCR, with quality assessment using a human gene. PC and NC paired nasal and saliva swab samples were collected from 91 and 92 children, respectively. Performance and interrater agreement (Cohen's κ) of PC versus NC nasal swab samples for viruses combined showed sensitivity of 91.6% (95% confidence interval [CI], 85.47 to 95.73%) and κ of 0.84 (95% CI, 0.79 to 0.88), respectively; the respective values for bacteria combined were 91.4% (95% CI, 86.85 to 94.87%) and κ of 0.85 (95% CI, 0.80 to 0.89). In saliva samples, viral and bacterial sensitivities were lower at 69.0% (95% CI, 57.47 to 79.76%) and 78.1% (95% CI, 71.60 to 83.76%), as were κ values at 0.64 (95% CI, 0.53 to 0.72) and 0.70 (95% CI, 0.65 to 0.76), respectively. Quality assessment for human biological material (18S rRNA) indicated perfect interrater agreement. At-home PC nasal swab samples performed comparably to NC swab samples, whereas PC saliva swab samples lacked sensitivity for the detection of respiratory microbes. IMPORTANCE RTIs are ubiquitous among children. Diagnosis involves a swab sample being taken by a health professional, which places a considerable burden on community health care systems, given the number of cases involved. The coronavirus disease 2019 (COVID-19) pandemic has seen an increase in the at-home self-collection of upper respiratory tract swab samples without the involvement of health professionals. It is advised that parents conduct or supervise swabbing of children. Surprisingly, few studies have addressed the quality of PC swab samples for subsequent identification of respiratory pathogens. We compared NC and PC nasal and saliva swab samples taken from the same child with RTI symptoms, for detection of respiratory pathogens. The PC nasal swab samples performed comparably to NC samples, whereas saliva swab samples lacked sensitivity for the detection of respiratory microbes. Collection of swab samples by parents would greatly reduce the burden on community nurses without reducing the effectiveness of diagnoses.

A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA.

Influenza A virus segment 2 is known to encode two polypeptides in overlapping open reading frames: PB1, the polymerase, and PB1-F2, a proapoptotic virulence factor. We show that a third major polypeptide is synthesized from PB1 mRNA via differential AUG codon usage. PB1 codon 40 directs translation of an N-terminally truncated version of the polypeptide (N40) that lacks transcriptase function but nevertheless interacts with PB2 and the polymerase complex in the cellular environment. Importantly, the expression of N40, PB1-F2, and PB1 are interdependent, and certain mutations previously used to ablate PB1-F2 production affected N40 accumulation. Removal of the PB1-F2 AUG upregulated N40 synthesis, while truncating PB1-F2 after codon 8 (with a concomitant M40I change in PB1) abolished N40 expression. A virus lacking both N40 and PB1-F2 replicated normally. However, viruses that did not express N40 but retained an intact PB1-F2 gene overexpressed PB1 early in infection and replicated slowly in tissue culture. Thus, the influenza A virus proteome includes a 12th primary translation product that (similarly to PB1-F2) is nonessential for virus viability but whose loss, in particular genetic backgrounds, is detrimental to virus replication.

Primary care clinicians' views of paediatric respiratory infection surveillance information to inform clinical decision-making: a qualitative study.

AIM: To investigate primary care clinicians' views of a prototype locally relevant, real-time viral surveillance system to assist diagnostic decision-making and antibiotic prescribing for paediatric respiratory tract infections (RTI). Clinicians' perspectives on the content, anticipated use and impact were explored to inform intervention development. BACKGROUND: Children with RTIs are overprescribed antibiotics. Pressures on primary care and diagnostic uncertainty can lead to decisional biases towards prescribing. We hypothesise that real-time paediatric RTI surveillance data could reduce diagnostic uncertainty and help reduce unnecessary antibiotic prescribing. METHODOLOGY: Semistructured one-to-one interviews with 21 clinicians from a range of urban general practitioner surgeries explored the clinical context and views of the prototype system. Transcripts were analysed using thematic analysis. RESULTS: Though clinicians self-identified as rational (not over)prescribers, cognitive biases influenced antibiotic prescribing decisions. Clinicians sought to avoid 'anticipated regret' around not prescribing for a child who then deteriorated. Clinicians were not aware of formal infection surveillance information sources (tending to assume many viruses are around), perceiving the information as novel and potentially useful. Perceptions of surveillance information as presented included: not relevant to decision-making/management; useful to confirm decisions post hoc; and increasing risks of missing sick children. Clinicians expressed wariness of using population-level data to influence individual patient decision-making and expressed preference for threat (high-risk) information identified by surveillance, rather than reassuring information about viral RTIs. CONCLUSIONS: More work is needed to develop a surveillance intervention if it is to beneficially influence decision-making and antibiotic prescribing in primary care. Key challenges for developing interventions are how to address cognitive biases and how to communicate reassuring information to risk-oriented clinicians.

Population-based paediatric respiratory infection surveillance: a prospective inception feasibility cohort study.

BACKGROUND: There is a need to reduce unnecessary general practitioner (GP) consultations and improve antibiotic stewardship in primary care. Respiratory tract infections (RTIs) in children are the most common reason for consulting and prescribing. Most RTI research is conducted at the point of consultation, leaving a knowledge gap regarding the population burden of RTIs. METHODS: Community-based, online prospective inception cohort study with nested qualitative study, to evaluate the feasibility and acceptability of collecting RTI symptom and microbiological data from children recruited prior to RTI onset. RESULTS: Parents of 10,310 children were invited. Three hundred thirty-one parents of 485 (4.7%) children responded and completed baseline data. Respondents were less socioeconomically deprived (p < 0.001) with younger (median ages 4 vs. 6 years, p < 0.001) children than non-respondents. The same parents reported 346 RTI episodes in 259 children, and 305 RTIs (in 225 children) were retained to parent-reported symptom resolution. Restricting analyses to the first RTI episode per family (to account for clustering effects), parents fully completed symptom diaries for 180 (87%) of 192 first illness episodes. Research nurses conducted home visits for 199 RTI episodes, collecting complete (symptomatic) swab sets in 195 (98%). Parents collected 194 (98% of 199 possible) symptomatic (during the nurse visit) and 282 (92% of 305 possible) asymptomatic swab sets (on symptom resolution, no nurse present). Interviews with 30 mothers and 11 children indicated study acceptability. CONCLUSIONS: Invitation response rates were in the expected range. The high retention and qualitative evidence suggest that community-based paediatric syndromic and microbiological surveillance research is feasible.

Nicotine preloading for smoking cessation: the Preloading RCT.

BACKGROUND: Nicotine preloading means using nicotine replacement therapy prior to a quit date while smoking normally. The aim is to reduce the drive to smoke, thereby reducing cravings for smoking after quit day, which are the main cause of early relapse. A prior systematic review showed inconclusive and heterogeneous evidence that preloading was effective and little evidence of the mechanism of action, with no cost-effectiveness data. OBJECTIVES: To assess (1) the effectiveness, safety and tolerability of nicotine preloading in a routine NHS setting relative to usual care, (2) the mechanisms of the action of preloading and (3) the cost-effectiveness of preloading. DESIGN: Open-label randomised controlled trial with examination of mediation and a cost-effectiveness analysis. SETTING: NHS smoking cessation clinics. PARTICIPANTS: People seeking help to stop smoking. INTERVENTIONS: Nicotine preloading comprised wearing a 21 mg/24 hour nicotine patch for 4 weeks prior to quit date. In addition, minimal behavioural support was provided to explain the intervention rationale and to support adherence. In the comparator group, participants received equivalent behavioural support. Randomisation was stratified by centre and concealed from investigators. MAIN OUTCOME MEASURES: The primary outcome was 6-month prolonged abstinence assessed using the Russell Standard. The secondary outcomes were 4-week and 12-month abstinence. Adverse events (AEs) were assessed from baseline to 1 week after quit day. In a planned analysis, we adjusted for the use of varenicline (Champix®; Pfizer Inc., New York, NY, USA) as post-cessation medication. Cost-effectiveness analysis took a health-service perspective. The within-trial analysis assessed health-service costs during the 13 months of trial enrolment relative to the previous 6 months comparing trial arms. The base case was based on multiple imputation for missing cost data. We modelled long-term health outcomes of smoking-related diseases using the European-study on Quantifying Utility of Investment in Protection from Tobacco (EQUIPT) model. RESULTS: In total, 1792 people were eligible and were enrolled in the study, with 893 randomised to the control group and 899 randomised to the intervention group. In the intervention group, 49 (5.5%) people discontinued preloading prematurely and most others used it daily. The primary outcome, biochemically validated 6-month abstinence, was achieved by 157 (17.5%) people in the intervention group and 129 (14.4%) people in the control group, a difference of 3.02 percentage points [95% confidence interval (CI) -0.37 to 6.41 percentage points; odds ratio (OR) 1.25, 95% CI 0.97 to 1.62; p = 0.081]. Adjusted for use of post-quit day varenicline, the OR was 1.34 (95% CI 1.03 to 1.73; p = 0.028). Secondary abstinence outcomes were similar. The OR for the occurrence of serious AEs was 1.12 (95% CI 0.42 to 3.03). Moderate-severity nausea occurred in an additional 4% of the preloading group compared with the control group. There was evidence that reduced urges to smoke and reduced smoke inhalation mediated the effect of preloading on abstinence. The incremental cost-effectiveness ratio at the 6-month follow-up for preloading relative to control was £710 (95% CI -£13,674 to £23,205), but preloading was dominant at 12 months and in the long term, with an 80% probability that it is cost saving. LIMITATIONS: The open-label design could partially account for the mediation results. Outcome assessment could not be blinded but was biochemically verified. CONCLUSIONS: Use of nicotine-patch preloading for 4 weeks prior to attempting to stop smoking can increase the proportion of people who stop successfully, but its benefit is undermined because it reduces the use of varenicline after preloading. If this latter effect could be overcome, then nicotine preloading appears to improve health and reduce health-service costs in the long term. Future work should determine how to ensure that people using nicotine preloading opt to use varenicline as cessation medication. TRIAL REGISTRATION: Current Controlled Trials ISRCTN33031001. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 41. See the NIHR Journals Library website for further project information.

Parent views on the content and potential impact of respiratory tract infection surveillance information: semistructured interviews to inform future research.

OBJECTIVES: This study explored the potential value of real-time information regarding respiratory tract infections (RTIs) circulating in the community by eliciting parent views on illustrative surveillance information and its possible impact on primary care consultations. DESIGN: Semistructured interviews were conducted with parents of children (>3 months-15 years). Participants were presented with example information on circulating viruses, symptoms and symptom duration and asked about its potential impact on perceptions of child illness and management practices. Interviews were analysed using the framework method. SETTING: Parents participating in a cohort study were selected purposefully using index of multiple deprivation and child age. PARTICIPANTS: 30 mothers of children (>3 months-15years). RESULTS: Parents anticipated using the information to inform lay diagnoses particularly when child symptoms were severe and thought normal symptom duration awareness might extend the time prior to seeking medical advice, but it also may encourage consultations when symptoms exceed the given duration. The information was not expected to change consultation behaviour if parents felt their child needed a medical evaluation and they felt unable to manage the symptoms. Most parents felt that the information could provide reassurance that could reduce intention to consult, but some felt it could raise concerns, by heightening awareness of circulating viruses. Lastly, parents wanted advice about protecting children from circulating viruses and felt that general practitioners using the information to diagnose child RTIs with greater certainty was acceptable. CONCLUSIONS: Diverse responses to the surveillance information were elicited, and there was some support for the intended outcomes. This study has important implications for the design of interventions to modify consulting behaviour. Future piloting to measure behaviour change in response to infection surveillance information are needed.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

Community paediatric respiratory infection surveillance study protocol: a feasibility, prospective inception cohort study.

INTRODUCTION: Paediatric respiratory tract infections (RTIs) are common reasons for primary care consultations and antibiotic prescribing. Locally relevant syndromic and microbiological surveillance information has the potential to improve the care of children with RTIs by normalising illness (parents) and reducing uncertainty (clinicians). Currently, most RTI studies are conducted at the point of healthcare service consultation, leaving the community burden, microbiology, symptom duration and proportion consulting largely unknown. This study seeks to establish the feasibility of (mainly online) participant recruitment and retention, and the acceptability/comparability of parent versus nurse-collected microbiological sampling, to inform the design of a future surveillance intervention study. Evidence regarding consultation rates and symptom duration is also sought. METHODS AND ANALYSIS: A community-based, feasibility prospective inception cohort study, recruiting children aged ≥3 months and <16 years and their parents via general practitioner surgery invitation letter, aiming to collect data on 300 incident RTIs by July 2016. Following informed consent, parents provide baseline (demographic) data online, and respond to weekly emails to confirm the absence/presence of new RTI symptoms. Once symptomatic, parents provide daily data online (RTI symptoms, school/day-care attendance, time off work, health service use, medication), and a research nurse visits to collect clinical examination data and microbiological (nasal and saliva) swabs. Parents are invited to provide symptomatic (at nurse visit, but without nurse assistance) and asymptomatic (alone) swabs on recovery. A review of primary care medical notes will gather medical history, health service utilisation, referral and antibiotic prescribing rates. Feasibility will be assessed using recruitment and retention rates, data completeness; and acceptability by quantitative survey and qualitative interviews. Symptomatic parent and nurse swab pairs will be compared for microbe isolation.

A new role for clathrin adaptor proteins 1 and 3 in lipoplex trafficking.

Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes.

Rev regulates translation of human immunodeficiency virus type 1 RNAs.

Full-length human immunodeficiency virus type 1 (HIV-1) RNA acts as both mRNA, encoding Gag and Gag-Pol polyproteins, and genomic RNA. Translation of this RNA must be tightly controlled to allow sufficient protein synthesis prior to a switch to particle production. The viral protein Rev stimulates nuclear export of unspliced HIV-1 RNAs containing the Rev response element, but may also stimulate translation of these RNAs. We previously identified an additional Rev binding site in the 5' untranslated region of the HIV-1 RNA. We show that Rev inhibits translation non-specifically at high concentrations and stimulates translation of HIV-1 RNAs at intermediate concentrations in vitro. Stimulation is dependent on the presence of the Rev binding site within the 5' untranslated region and not on the Rev response element. In COS-1 cells, translation from an HIV-1 reporter is specifically increased by coexpression of Rev.

Internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of Unr to two distinct sites on the 5' untranslated region.

Internal initiation of translation from the human rhinovirus-2 (HRV-2) internal ribosome entry site (IRES) is dependent upon host cell trans-acting factors. The multiple cold shock domain protein Unr and the polypyrimidine tract-binding protein have been identified as synergistic activators of HRV-2 IRES-driven translation. In order to investigate the mechanism by which Unr acts in this process, we have mapped the binding sites of Unr to two distinct secondary structure domains of the HRV-2 IRES, and have identified specific nucleotides that are involved in the binding of Unr to the IRES. The data suggest that Unr acts as an RNA chaperone to maintain a complex tertiary IRES structure required for translational competency.

Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation.

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.