Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-Related Quality Attributes in Protein Therapeutics.

In vitro models that mimic the in vivo environment can greatly facilitate and support criticality assessment of product quality attributes for therapeutic drugs to ensure product quality. An in vitro model is established to study and predict the impact of thiol-related attributes on safety or efficacy of intraocular antibody products. This model simulates the physiological redox environment of rabbit vitreous and maintains a steady-state redox potential using reduced and oxidized forms of glutathione. A similar in vitro model that mimics the thiol redox conditions of human blood has been previously established and has become a predictive tool to study intravenous (IV) therapeutic proteins. We utilized both vitreous and serum models to study the potential impact of antibody variants (trisulfides and free-thiols) on product qualities of different antibodies. The studies demonstrate that both models are effective tools to monitor changes of thiol-related attributes under physiological conditions, providing insights on these thiol-related attributes and allowing for more informed assessment of biological relevance and criticality of the attributes. Furthermore, we propose that the approach using an in vitro study for the product quality attribute assessment can be used to predict in vivo effects for future molecules during the development of biopharmaceuticals, reducing the need for live subject studies.

Streamlined Characterization of an Antibody-Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry.

This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody-drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5-3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Trisulfide modification impacts the reduction step in antibody-drug conjugation process.

Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.

A novel population of myeloid cells responding to coxsackievirus infection assists in the dissemination of virus within the neonatal CNS.

Enterovirus infection in newborn infants is a significant cause of aseptic meningitis and encephalitis. Using a neonatal mouse model, we previously determined that coxsackievirus B3 (CVB3) preferentially targets proliferating neural stem cells located in the subventricular zone within 24 h after infection. At later time points, immature neuroblasts, and eventually mature neurons, were infected as determined by expression of high levels of viral protein. Here, we show that blood-derived Mac3(+) mononuclear cells were rapidly recruited to the CNS within 12 h after intracranial infection with CVB3. These cells displayed a myeloid-like morphology, were of a peripheral origin based on green fluorescent protein (GFP)-tagged adoptive cell transplant examination, and were highly susceptible to CVB3 infection during their migration into the CNS. Serial immunofluorescence images suggested that the myeloid cells enter the CNS via the choroid plexus, and that they may be infected during their extravasation and passage through the choroid plexus epithelium; these infected myeloid cells ultimately penetrate into the parenchyma of the brain. Before their migration through the ependymal cell layer, a subset of these infected myeloid cells expressed detectable levels of nestin, a marker for neural stem and progenitor cells. As these nestin(+) myeloid cells infected with CVB3 migrated through the ependymal cell layer, they revealed distinct morphological characteristics typical of type B neural stem cells. The recruitment of these novel myeloid cells may be specifically set in motion by the induction of a unique chemokine profile in the CNS induced very early after CVB3 infection, which includes upregulation of CCL12. We propose that intracranial CVB3 infection may lead to the recruitment of nestin(+) myeloid cells into the CNS which might represent an intrinsic host CNS repair response. In turn, the proliferative and metabolic status of recruited myeloid cells may render them attractive targets for CVB3 infection. Moreover, the migratory ability of these myeloid cells may point to a productive method of virus dissemination within the CNS.

Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.

Coxsackieviruses are significant human pathogens, and the neonatal central nervous system (CNS) is a major target for infection. Despite the extreme susceptibility of newborn infants to coxsackievirus infection and viral tropism for the CNS, few studies have been aimed at determining the long-term consequences of infection on the developing CNS. We previously described a neonatal mouse model of coxsackievirus B3 (CVB3) infection and determined that proliferating stem cells in the CNS were preferentially targeted. Here, we describe later stages of infection, the ensuing inflammatory response, and subsequent lesions which remain in the adult CNS of surviving animals. High levels of type I interferons and chemokines (in particular MCP-5, IP10, and RANTES) were upregulated following infection and remained at high levels up to day 10 postinfection (p.i). Chronic inflammation and lesions were observed in the hippocampus and cortex of surviving mice for up to 9 months p.i. CVB3 RNA was detected in the CNS up to 3 months p.i at high abundance ( approximately 10(6) genomes/mouse brain), and viral genomic material remained detectable in culture after two rounds of in vitro passage. These data suggest that CVB3 may persist in the CNS as a low-level, noncytolytic infection, causing ongoing inflammatory lesions. Thus, the effects of a relatively common infection during the neonatal period may be long lasting, and the prognosis for newborn infants recovering from acute infection should be reexplored.

Host and virus determinants of picornavirus pathogenesis and tropism.

The family Picornaviridae contains some notable members, including rhinovirus, which infects humans more frequently than any other virus; poliovirus, which has paralysed or killed millions over the years; and foot-and-mouth-disease virus, which led to the creation of dedicated institutes throughout the world. Despite their profound impact on human and animal health, the factors that regulate pathogenesis and tissue tropism are poorly understood. In this article, we review the clinical and economic challenges that these agents pose, summarize current knowledge of host-pathogen interactions and highlight a few of the many outstanding questions that remain to be answered.

A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide.

A robust, high-throughput method using hydrophilic interaction liquid chromatography (HILIC) coupled with a charged aerosol detector (CAD) is reported as a novel approach for trisulfide quantitation in monoclonal antibodies (mAbs). The products of mAb reduction using tris(2-carboxyethyl)phosphine (TCEP) include a species (TCEP(S)) that is stoichiometrically produced from trisulfides. The TCEP reaction products are chromatographically separated, detected, and quantified by the HILICCAD method. The method was qualified to quantify trisulfides across a range of 1-40% (mol trisulfide/mol mAb). In all tested matrix components, assay linearity and intermediate precision were established with correlation coefficients (R(2))>0.99, and relative standard deviations (RSD)<10%. A method comparability study was performed using peptide mapping LC-MS as an orthogonal measurement. For the range of 1-40% trisulfides, the analysis demonstrates that, on average, HILICCAD reads between 0.95 and 1.10 times the value of LC-MS with 95% confidence. Applications of the HILICCAD method include trisulfide determination in purified mAbs to be used in the production of cysteine-linked antibody-drug conjugates, and in cell culture development studies to understand sources of, and strategies for control of, trisulfides.

Weight loss through adjustable gastric banding and improvement in daytime sleepiness: 2 year interim results of APEX study.

OBJECTIVE: Obesity is one factor associated with an increased risk of obstructive sleep apnea (OSA). This study reports the investigator-reported resolution or improvement of OSA and improvements in sleep-related quality of life (QOL) 2 years after surgical placement of the LAP-BAND AP * (LBAP) system. RESEARCH DESIGN AND METHODS: The LBAP Experience (APEX) study is an ongoing 5 year, prospective, observational study assessing change in weight, comorbidities, and QOL after LBAP implantation. This is an interim analysis of patients with evaluable data at 24 months who had OSA at baseline. CLINICAL TRIAL REGISTRATION: NCT00501085. RESULTS: At baseline, 117 of 395 patients (29.6%; mean body mass index [BMI], 45.0 kg/m2) reported OSA; of these, 57 had evaluable patient-reported outcome data at 2 years. Investigator-reported resolution or improvement of OSA was 69% and 86% at post-operative years 1 and 2, respectively. Patients reporting resolution, improvement, or no change in OSA experienced mean changes in BMI and percentage of weight loss of -9.7 kg/m(2)/-21.7%, -8.3 kg/m(2)/-18.7%, and -5.7 kg/m(2)/-13.2%, respectively (n = 54). Mean 2 year BMI was not statistically different between the groups (p = not significant). Mean scores for all Epworth Sleepiness Scale responses for the OSA population improved by -0.43 from baseline (p < 0.0001; n = 78) compared with -0.29 for patients without OSA at baseline (n = 177; p = 0.037 between groups). In addition, the overall study population experienced resolution and/or improvement in other obesity-related comorbidities, such as type 2 diabetes (96%) and hypertension (91%). CONCLUSIONS: These data support that surgically facilitated weight loss can improve sleep-related QOL and may result in resolution or improvement of OSA; the degree of weight loss may be related to these changes.

Efficacy and safety of the adjustable gastric band - pooled interim analysis of the APEX and HERO studies at 48 weeks.

OBJECTIVE: This 48 week combined analysis reports safety and clinical effectiveness of the LAP-BAND AP * laparoscopic adjustable gastric band (LAGB) in severely obese patients enrolled in the 5 year, prospective, observational, open-label APEX (NCT00501085) and HERO (NCT00953173) studies. METHODS: The studies enrolled 1620 patients (APEX: N = 514; HERO: N = 1106), 1140 patients in the US (including all APEX patients), and 480 patients in the European Union (EU), Canada or Australia. APEX and HERO are non-randomized, non-comparator, open-label studies with differences in study management practices and follow-up. Notably, laboratory data were not collected during the APEX study. RESULTS: After 48 weeks, mean (SD) percentage weight loss (%WL) was for APEX: 18.7% (7.9); HERO-US: 17.9% (8.5); HERO-EU: 16.5% (10.3); HERO-Canada: 13.4% (8.9); and HERO-Australia: 12.3% (6.9). After 48 weeks, there were no significant differences in %WL for APEX vs. HERO-US. After 48 weeks in the combined analysis (APEX + HERO): (1) patients without vs. with type 2 diabetes at baseline had greater %WL (18% [8.7] vs. 16% [8.5], p = 0.002); (2) female patients had greater %WL vs. male patients (17.9% [8.5] vs. 15.9% [9.3], p = 0.003); (3) younger patients had greater %WL vs. older patients (<50 years: 17.8% [8.7] vs. ≥50 years: 16.7% [8.6], p = 0.035); (4) baseline BMI did not affect %WL (≤35 to ≤45 kg/m(2): 17.7% [8.4] vs. >45 kg/m(2): 17.1% [9.1], p = 0.272). Device-related serious adverse events and adverse events were reported in 1.9% and 17.7% of patients, respectively. Revision and explantation surgeries were carried out on 3.4% and 2.3% of patients, respectively during the 48 weeks of follow-up. CONCLUSIONS: This analysis demonstrates the effective weight loss and safety profile of the current LAGB system, with US patients achieving better weight loss than patients from outside the US.

Quality of life after laparoscopic adjustable gastric banding (LAP-BAND): APEX interim 3-year analysis.

BACKGROUND: Obesity is often associated with diminished health-related quality of life (HRQOL), but significant gains in HRQOL have been observed after bariatric surgery. Laparoscopic adjustable gastric banding has been established as a safe, effective treatment to reduce weight in patients with obesity. This report summarizes interim 3-year changes in HRQOL and body weight, as well as safety postimplantation of the LAP-BAND AP (LBAP) system. METHODS: The LAP-BAND AP EXperience (APEX) trial, an ongoing, prospective, 5-year, open-label study, assessed changes in HRQOL (Obesity and Weight-Loss Quality of Life [OWLQOL] questionnaire) and body weight, and safety after placement of LBAP. This interim analysis represents patients with evaluable OWLQOL data at baseline and at 3 years (n = 183). RESULTS: The OWLQOL total score and individual scores significantly improved within 6 months post-LBAP and continued to improve during a 3 year period (P < 0.0001, both). Total score improved from 71.0 to 34.0 (mean improvement from baseline, 52%; range, 18%-65%); mean change in individual scores was -2.2 (range, -0.7 to -3.0). Percent weight loss was maintained through 3 years (19.4%; n = 174). Improvement in OWLQOL was associated with percent weight loss at 3 years (r = -0.5407; P < 0.0001). Revisions and explants were performed in 7 (3.8%) and 20 (10.9%) out of 183 patients, respectively. CONCLUSIONS: Meaningful improvement in quality of life occurred through clinically significant weight loss after LBAP placement, extending throughout the 3 years of this analysis.

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20.

Polysorbate 20 (PS-20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS-20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS-20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS-20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS-20 was measured by a mixed-mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS-20 was characterized by (1) H-NMR and a reverse-phase chromatography method. Non-specific adsorption of PS-20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS-20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS-20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di-esters and tri-esters.

Effect of early weight loss on type 2 diabetes mellitus after 2 years of gastric banding.

OBJECTIVE: Laparoscopic adjustable gastric banding (LAGB) is an established bariatric surgical procedure that produces meaningful weight loss and improvements in patients with type 2 diabetes mellitus (T2DM) and a body mass index (BMI) ≥ 30 kg/m2. This study examined the effect of LAGB on T2DM status in severely obese patients with T2DM. METHODS: This was a 2-year interim analysis of patients with T2DM who required daily hypoglycemic medication at baseline (N = 89) in the 5-year, open-label, prospective, observational LAP-BAND AP® EXperience (APEX) trial. Type 2 diabetes mellitus status was classified as "remission," "improved," "stable," or "worse" based on physician- and patient-reported changes in T2DM control and changes in hypoglycemic medication use. RESULTS: At baseline, 89 (22.5%) of 395 patients required daily hypoglycemic medication; 66 patients had data available after 2 years. Remission of T2DM occurred in 32 (48.5%) patients, improvement occurred in 31 (47.0%) patients, and no change occurred in 3 (4.5%) patients. Overall, 95.5% of patients experienced remission or improvement in T2DM status. Duration of T2DM in patients with remission or improvement after 2 years was 4.0 and 6.7 years, respectively (P = 0.082 between groups), and was associated with change in T2DM status (logistic regression, P = 0.069). Baseline BMI, change in BMI, and percent weight loss were not significantly different between the T2DM response groups. Percent excess weight loss was numerically, but not statistically significantly, greater in remitted (-56.1%) compared with the improved response (-42.9%) group (P = 0.134), and was correlated with change in T2DM status (logistic regression, P = 0.052). After 2 years, patients experienced remission or improvement of other obesity comorbidities. The rate of revisional surgery or explantation was 3.4%, and it was not significantly different between patients with and without T2DM (P = 0.687). CONCLUSION: Shorter duration of T2DM and greater percent excess weight loss were associated with an increased likelihood of remission or improvement in T2DM status through LAGB after 2 years. Laparoscopic adjustable gastric banding is a potential adjunctive treatment for obese patients with T2DM.

Safety and efficacy of the LAP-BAND AP® adjustable gastric band in the treatment of obesity: results at 2 years.

BACKGROUND: The objective of this analysis is to report interim, 2-year results for morbidly obese patients who have undergone laparoscopic adjustable gastric banding in the LAP-BAND AP® EXperience (APEX) trial. METHODS: The APEX trial is an ongoing, multicenter, prospective, open-label, 5-year study of the LAP-BAND AP® System (LBAP) in 517 morbidly obese patients at 50 clinical centers in the United States. Last observation carried forward was used in the analyses of change in body weight and comorbid conditions, and observed data were analyzed for the Obesity and Weight-Loss Quality of Life (OWLQOL) questionnaire. Changes in body weight, percent weight loss, percent excess weight loss, body mass index (BMI), OWLQOL score, remission or improvement in obesity-related comorbid conditions, and adverse events were reported. RESULTS: At baseline, 81.5% of patients were female, and 85.8% were white. The mean age was 42.5 years, and the mean BMI was 44.0 kg/m(2). More than 85% of patients had ≥ 1 obesity-related comorbidity. At 2 years, the mean BMI change was -8.5 kg/m(2), and the mean percent weight loss was -19.3%. Responses to all questions on the OWLQOL questionnaire had a mean improvement of 54% (range, 26%-67%) at 2 years. Obesity-related comorbid conditions were remitted or improved in the majority of patients at 2 years, including type 2 diabetes mellitus (96%), hypertension (91%), gastroesophageal reflux disease (91%), hyperlipidemia (77%), obstructive sleep apnea (86%), depression (75%), and osteoarthritis (93%). The LBAP and its implantation were well tolerated, with 19.1% and 6.0% of patients reporting device-related adverse events or serious device-related adverse events, respectively. CONCLUSION: The LBAP safely and effectively facilitated weight loss in morbidly obese patients, with clinically meaningful improvements in quality of life and obesity-related comorbidities. The durability of these results will be further described with additional follow-up through 5 years. TRIAL REGISTRATION: www.ClinicalTrials.gov identifier NCT00501085.

Effect of adjustable gastric banding on changes in gastroesophageal reflux disease (GERD) and quality of life.

OBJECTIVE: Bariatric surgery is an effective treatment for the reduction of weight in obese patients (BMI ≥ 40 kg/m(2) or 30 kg/m(2) with ≥1 comorbidities), who are refractory to behavioral and medical therapies. This study examined the effect of the adjustable gastric band (AGB) system on changes in gastroesophageal reflux disease (GERD) and patient-reported outcomes, including measures of quality of life. METHODS: Two-year interim analysis of patients (N = 171) in the 5 year, prospective APEX study who reported GERD prior to the AGB procedure. An unrecorded number of hiatal hernia repairs were conducted during the APEX study. RESULTS: At baseline, 171 of 395 patients (43%) reported GERD requiring daily medical therapy. After 2 years, 122 patients had sufficient data to assess outcome (71%). Complete resolution of GERD was reported in 98 patients (80%), improvement in 13 (11%), no change in 9 (7%), and worsening in 2 (2%). Overall, 91% of GERD patients experienced resolution and/or improvement of GERD. Baseline BMI was not significantly different among the GERD response categories (resolved, improved, and stable/worse), p = 0.4581. Mean ΔBMI and percentage excess weight loss (%EWL) were: -8.8 kg/m(2)/-0.9%, -11.4 kg/m(2)/-53.9%, -6.4 kg/m(2)/-36.1%, and -7.1 kg/m(2)/-31.2%, respectively. There were no significant differences in reductions in BMI or %EWL between responder groups (resolved versus stable/worse ΔBMI p = 0.1031, %EWL p = 0.0667 OR resolved/improved versus stable/worse ΔBMI p = 0.0918, %EWL p = 0.0552). After 2 years, resolution or improvement occurred in pre-existing comorbidities: type 2 diabetes (96%), hypertension (91%), hyperlipidemia (77%), obstructive sleep apnea (86%), osteoarthritis (93%), and depression (75%). Patient satisfaction with AGB was assessed as: very satisfied/satisfied (87%), very satisfied (50%), dissatisfied (5.0%). Quality of life measured by the Obesity and Weight-Loss Quality of Life Instrument (GERD patients) significantly improved from baseline. CONCLUSION: Obese patients with GERD had meaningful improvement in patient-reported outcomes with the AGB system. In addition, other obesity-related comorbidities and measures of quality of life improved.

Deletions within the 5'UTR of coxsackievirus B3: consequences for virus translation and replication.

Key features of an ideal RNA-based vaccine against coxsackievirus B3 (CVB3) are (i) limited genome replication/virus production (to minimize vaccine-related pathology) and (ii) abundant virus protein synthesis (to maximize immunogenicity). These attributes may apply to CVB3 RNAs lacking up to 250 nucleotides (nt) from their 5' terminus; these RNAs do not give rise to infectious progeny, but they have been reported to retain the entire CVB3 IRES (mapped to nt approximately 432-639) and to produce large quantities of viral protein in transfected cells. Here, we constructed five 5' RNA deletion variants that, to our surprise, failed to protect against CVB3 challenge. We investigated the reasons for this failure and conclude that (i) a 5' terminal deletion as short as 32 nt abolishes CVB3 RNA replication in transfected cells; (ii) this deleted RNA, and others with longer deletions, do not direct abundant protein synthesis in transfected cells, probably as a consequence of their replicative incapacity; and (iii) the CVB3 IRES is substantially larger than previously thought, and its 5' boundary lies between residues 76 and 125, very closely approximating that of the poliovirus IRES.

Coxsackievirus B3 proteins directionally complement each other to downregulate surface major histocompatibility complex class I.

Picornaviruses carry a small number of proteins with diverse functions that subvert and exploit the host cell. We have previously shown that three coxsackievirus B3 (CVB3) proteins (2B, 2BC, and 3A) target the Golgi complex and inhibit protein transit. Here we investigate these effects in more detail and evaluate the distribution of major histocompatibility complex (MHC) class I molecules, which are critical mediators of the CD8(+) T-cell response. We report that concomitant with viral protein synthesis, MHC class I surface expression is rapidly downregulated during infection. However, this phenomenon may not result solely from inhibition of anterograde trafficking; we propose a new mechanism whereby the CVB3 2B and 2BC proteins upregulate the internalization of MHC class I (and possibly other surface proteins), perhaps by focusing of endocytic vesicles at the Golgi complex. Thus, our findings indicate that CVB3 carries at least three nonstructural proteins that directionally complement one another; 3A disrupts the Golgi complex to inhibit anterograde transport, while 2B and/or 2BC upregulates endocytosis, rapidly removing proteins from the cell surface. Taken together, these effects may render CVB3-infected cells invisible to CD8(+) T cells and untouchable by many antiviral effector molecules. This has important implications for immune evasion by CVB3.

Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle.

Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.

Analysis of translational initiation in coxsackievirus B3 suggests an alternative explanation for the high frequency of R+4 in the eukaryotic consensus motif.

Translational initiation of most eukaryotic mRNAs occurs when a preinitiation complex binds to the 5' cap, scans the mRNA, and selects a particular AUG codon as the initiation site. Selection of the correct initiation codon relies, in part, on its flanking residues; in mammalian cells, the core of the "Kozak" consensus is R-3CCAUGG+4 (R=purine; the A residue is designated position +1). The R-3 is considered the most important flanking residue, followed by G+4. Picornaviral mRNAs differ from most cellular mRNAs in several ways; they are uncapped, and they contain an internal ribosome entry site that allows the ribosome to bind near the initiation codon. The initiation codon of coxsackievirus B3 (CVB3) is flanked by both R-3 and G+4 (AAAATGG). Here, we report the construction of full-length CVB3 genomes that vary at these two positions, and we evaluate the effects of these variant sequences in vitro, in tissue culture cells, and in vivo. A virus with an A-->C transversion at position -3 replicates as well as wild-type CVB3, both in tissue culture and in vivo. This virus is highly pathogenic, and its sequence is stable throughout the course of an in vivo infection. Furthermore, the in vitro translation products from this RNA are very similar to the wild type. Thus, R-3-thought to be the most functionally important component of the Kozak consensus-appears to be dispensable in CVB3. In contrast, a G-to-C transversion at G+4 is lethal; RNAs carrying this mutation fail to generate infectious virus either in tissue culture or in vivo. However, in vitro analysis indicates that G+4 has only a marginal effect on translational initiation, especially if R-3 is present; instead, the G+4 is required mainly because the second triplet of the polyprotein open reading frame must encode glycine, without which infectious virus production cannot proceed. In summary, our data indicate that CVB3 remains viable, even in vivo, in the absence of R-3, and we propose that the most important factor contributing to the high frequency of G+4-not only in CVB but also in other eukaryotic mRNAs, and thus in the consensus motif itself-may be the constraint upon the second amino acid rather than the requirements for translational initiation.

An authentic 3' noncoding region is necessary for efficient poliovirus replication.

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).