scholarly journals Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization

2005 ◽  
Vol 385 (2) ◽  
pp. 363-370 ◽  
Author(s):  
Hannelore PETERS ◽  
Yuri Y. KUSOV ◽  
Sonja MEYER ◽  
Andrew J. BENIE ◽  
Englbert BÄUML ◽  
...  
Keyword(s):  
Life Cycle ◽  
Proteolytic Activity ◽  
Hepatitis A ◽  
Kinetic Resolution ◽  
Rna Binding ◽  
Hepatitis A Virus ◽  
Viral Life Cycle ◽  
Binding Kinetics ◽  
Viral Rna ◽  
Binding Studies

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24→Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (KD) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by kon and koff values of the order of 102 M−1·s−1 and 10−2 to 10−1 s−1 respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5′-terminal domain, followed fast binding kinetics with kon and koff values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a KD in the millimolar range, dimeric 3C had a significantly increased binding affinity with KD values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

scholarly journals Poly(rC)-binding protein 1 limits hepatitis C virus assembly and egress

2021 ◽  
Author(s):  
Sophie E. Cousineau ◽  
Selena M. Sagan
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Life Cycle ◽  
Viral Rna ◽  
Virion Assembly ◽  
Rna Accumulation

ABSTRACTThe hepatitis C virus (HCV) co-opts a number of cellular elements – including proteins, lipids, and microRNAs – to complete its viral life cycle. The cellular RNA-binding protein poly(rC)-binding protein 1 (PCBP1) had previously been reported to bind the HCV genome 5’ untranslated region (UTR), but its importance in the viral life cycle has remained unclear. Herein, we aimed to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that endogenous PCBP1 knockdown decreased viral RNA accumulation yet increased extracellular virus titers. To dissect PCBP1’s specific role in the viral life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, virion assembly and egress. We found that PCBP1 did not affect viral entry, translation, RNA stability, or RNA replication in the absence of efficient virion assembly. To specifically examine virion assembly and egress, we inhibited viral RNA replication with an RNA-dependent RNA polymerase inhibitor and tracked both intracellular and extracellular viral titers over time. We found that when viral RNA accumulation was inhibited, knockdown of PCBP1 still resulted in an overall increase in HCV particle secretion. We therefore propose a model where endogenous PCBP1 limits virion assembly and egress, thereby indirectly enhancing viral RNA accumulation in infected cells. This model furthers our understanding of how cellular RNA-binding proteins modulate HCV genomic RNA utilization during the viral life cycle.IMPORTANCEHepatitis C virus (HCV) is a positive-sense RNA virus, and as such, its genome must be a template for multiple mutually exclusive steps of the viral life cycle, namely translation, RNA replication, and virion assembly. However, the mechanism(s) that regulate how the viral genome is used throughout the viral life cycle still remain unclear. A cellular RNA-binding protein – PCBP1 – had previously been reported to bind the HCV genome, but its precise role in the viral life cycle was not known. In this study, we found that depleting PCBP1 decreased viral RNA accumulation but increased virus secretion. We ruled out a role for PCBP1 in virus entry, translation, genome stability or RNA replication, and demonstrate that PCBP1 knockdown enhances virus secretion when RNA replication is inhibited. We conclude that PCBP1 normally prevents virus assembly and egress, which allows more of the viral genomic RNA to be available for translation and viral RNA replication.

scholarly journals Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Asuka Hirai-Yuki ◽  
Lucinda Hensley ◽  
Jason K. Whitmire ◽  
Stanley M. Lemon
Keyword(s):  
Life Cycle ◽  
Bile Acids ◽  
Neutralizing Antibodies ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Acute Infection ◽  
Cell Culture Supernatant ◽  
Particle Type ◽  
Human Bile

ABSTRACTHepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replicationin vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infectedIfnar1−/−Ifngr1−/−andMavs−/−mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.IMPORTANCEHAV is a hepatotropic, fecally/orally transmitted picornavirus that can cause severe hepatitis in humans. Recent work reveals that it has an unusual life cycle. Virus is found in cell culture supernatant fluids in two mature, infectious forms: one wrapped in membranes (quasi-enveloped) and another that is nonenveloped. Membrane-wrapped virions circulate in blood during acute infection and are resistant to neutralizing antibodies, likely facilitating HAV dissemination within the liver. On the other hand, virus shed in feces is nonenveloped and highly stable, facilitating epidemic spread and transmission to naive hosts. Factors controlling the biogenesis of these two distinct forms of the virus in infected humans are not understood. Here we characterize vectorial release of quasi-enveloped virions from polarized epithelial cell cultures and provide evidence that bile acids strip membranes from eHAV following its secretion into the biliary tract. These results enhance our understanding of the life cycle of this unusual picornavirus.

Membrane association and RNA binding of recombinant hepatitis A virus protein 2C

1998 ◽  
Vol 143 (5) ◽  
pp. 931-944 ◽  
Author(s):  
Y. Y. Kusov ◽  
C. Probst ◽  
M. Jecht ◽  
P. D. Jost ◽  
V. Gauss-Müller
Keyword(s):  
Hepatitis A ◽  
Rna Binding ◽  
Hepatitis A Virus ◽  
Virus Protein ◽  
Membrane Association ◽  
Recombinant Hepatitis

scholarly journals Development of Methods To Detect “Norwalk-Like Viruses” (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak

2000 ◽  
Vol 66 (1) ◽  
pp. 213-218 ◽  
Author(s):  
Kellogg J. Schwab ◽  
Frederick H. Neill ◽  
Rebecca L. Fankhauser ◽  
Nicholas A. Daniels ◽  
Stephan S. Monroe ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Food Samples ◽  
Viral Rna ◽  
Norwalk Virus ◽  
Specific Primers ◽  
Food Borne ◽  
Common Causes ◽  
Food Borne Illness

ABSTRACT “Norwalk-like viruses” (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 102 to 103 viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

scholarly journals hnRNPA1 regulates early translation to replication switch in SARS-CoV-2 life cycle

2021 ◽  
Author(s):  
Ram Kumar ◽  
Yogesh Chander ◽  
Nitin Khandelwal ◽  
Himanshu Nagori ◽  
Assim Verma ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Binding ◽  
Life Forms ◽  
Viral Rna ◽  
Initiation Factor ◽  
Target Cells ◽  
Dependent Manner ◽  
Translational Initiation ◽  
Rna Methylation ◽  
Early Translation

ABSTRACTOur study suggests that methylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for its optimal replication in the target cells. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, an RNA-binding protein) was shown to mediate deposition of N6-methyladenosine (m6A) in internal SARS-CoV-2 RNA. The levels of hNRNPA1 expression and extent of methylation varied, depending on the course of SARS-CoV-2 life cycle. The recruitment of eIF4E (translational initiation factor) facilitated viral RNA translation at 1 hour post infection (1 hpi). However, at 2 hpi, methylation of internal SARS-CoV-2 RNA recruited hNRNPA1 which facilitated viral RNA transcription but resulted in translational repression, a phenomenon contributing in understanding the early translation to replication switch in the viral life cycle. Besides, the abrogation of methylation also produced a defective 5’ cap of viral RNA which failed to interact with eIF4E, thereby resulting in a decreased synthesis of viral proteins. To conclude, methylation of the internal and 5’ cap of SARS-CoV-2 RNA was shown to regulate transcription and translation of SARS-CoV-2 in a time dependent manner.IMPORTANCERNA modifications are found in all life forms and have been linked to development, health and diseases. Our study reveals that internal SARS-CoV-2 RNA methylation (m6A) is essential for interaction with hNRNPA1 to effectively synthesize viral genome. Besides, m6A-marked RNA and hRNPA1 interaction was also shown to regulate early translation to replication switch in SARS-CoV-2 life cycle. Blocking SARS-CoV-2 RNA methylation resulted in reduced virus yield, suggesting epitranscriptomic machinery (methylation) facilitates SARS-CoV-2 replication and might represent potential target for new antiviral drugs against COVID-19.

scholarly journals Proteolytic activity of hepatitis A virus 3C protein.

1991 ◽  
Vol 65 (5) ◽  
pp. 2595-2600 ◽  
Author(s):  
X Y Jia ◽  
E Ehrenfeld ◽  
D F Summers
Keyword(s):  
Proteolytic Activity ◽  
Hepatitis A ◽  
Hepatitis A Virus

scholarly journals Redundant Late Domain Functions of Tandem VP2 YPX3L Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus

2018 ◽  
Vol 92 (23) ◽  
Author(s):  
Olga González-López ◽  
Efraín E. Rivera-Serrano ◽  
Fengyu Hu ◽  
Lucinda Hensley ◽  
Kevin L. McKnight ◽  
...  
Keyword(s):  
Life Cycle ◽  
Capsid Protein ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Protein ◽  
Endosomal Sorting ◽  
Late Domain ◽  
Membrane Budding ◽  
Late Domains ◽  
Endosomal Vesicles

ABSTRACTThe quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.IMPORTANCENonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L “late domains” in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here, we describe YPX3L domain mutants that assemble capsids normally but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.

scholarly journals Detection of hepatitis A virus by extraction of viral RNA and molecular hybridization.

1987 ◽  
Vol 25 (10) ◽  
pp. 1822-1829 ◽  
Author(s):  
J R Ticehurst ◽  
S M Feinstone ◽  
T Chestnut ◽  
N C Tassopoulos ◽  
H Popper ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Viral Rna ◽  
Molecular Hybridization

scholarly journals Intracellular Hepatitis C Virus Modeling Predicts Infection Dynamics and Viral Protein Mechanisms

2018 ◽  
Vol 92 (11) ◽  
pp. e02098-17 ◽  
Author(s):  
Thomas R. Aunins ◽  
Katherine A. Marsh ◽  
Gitanjali Subramanya ◽  
Susan L. Uprichard ◽  
Alan S. Perelson ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Kinetic Data ◽  
Viral Proteins ◽  
Viral Life Cycle ◽  
Hcv Infection ◽  
Viral Rna ◽  
Model Parameters ◽  
Virus Life Cycle

ABSTRACTHepatitis C virus (HCV) infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these infections becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral life cycle. Here, we present a detailed mathematical model that represents the full hepatitis C virus life cycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses ofcis- andtrans-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady state. Our model predicts higher rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the life cycle.IMPORTANCEWe have designed a model for the full life cycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C virus replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded it to create a model that incorporates the full virus life cycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the life cycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study are a more physiologically relevant representation of a viral life cycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C virus infections.

scholarly journals Persistence of Hepatitis A Virus RNA in Water, on Non-porous Surfaces, and on Blueberries

2021 ◽  
Vol 12 ◽  
Author(s):  
Mathilde Trudel-Ferland ◽  
Eric Jubinville ◽  
Julie Jean
Keyword(s):  
Stainless Steel ◽  
Polyvinyl Chloride ◽  
Room Temperature ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Viral Rna ◽  
Gold Standard Method ◽  
Viral Particles ◽  
Initial Load ◽  
Phosphate Buffered Saline

Enteric viruses, such as human norovirus and hepatitis A virus (HAV), are the leading cause of transmissible foodborne illness. Fresh produce such as berries are often contaminated by infected food handlers, soiled water, or food contact surfaces. The gold-standard method for virus detection throughout the food chain is RT-qPCR, which detects portions of genomes including non-infectious viral particles and naked viral RNA. The aim of this study was to evaluate the persistence of heat-inactivated HAV in water, phosphate-buffered saline, on stainless steel and polyvinyl chloride, and on blueberries at −80°C, −20°C, 4°C, and room temperature. In water and phosphate-buffered saline, viral RNA could be detected for up to 90 days regardless of temperature when the initial load was 2.5 × 104 or 2.5 × 106 genome copies. It was detected on polyvinyl chloride and blueberries under most conditions. On stainless steel, the large initial load persisted for 90 days, while the medium-level load was detected only up to 16 days at room temperature or 60 days at 4°C. The detection of non-infectious viral RNA can confound investigations of gastroenteritis outbreaks. Pretreatments that discriminate between naked RNA, non-infectious virions and infectious virions need to be included in the RT-qPCR method in order to reduce the risk of positive results associated with non-infectious viral particles.

Sign in / Sign up

Export Citation Format

Share Document