Molecular Biology of the Flavivirus Chapter Abstracts
Chapter 1
The Molecular Biology of Yellow Fever Virus
Beate M. Kümmerer
Abstract
Like all the members of the family Flaviviridae, the yellow fever virus is an enveloped virus containing a single stranded RNA of positive polarity. After binding of the virion to the cell surface and receptor mediated endocytosis, the nucleocapsid is released into the cytoplasm where translation and RNA replication occurs. Translation results in a single polyprotein which is cleaved co- and posttranslationally by host cell and viral proteases to yield the viral proteins. The N-terminal part encodes the structural proteins C, prM and E, whereas the remaining two third C- terminally releases the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Specific functions have been attributed to most of the yellow fever viral proteins. Whereas the structural proteins are involved in virion formation, the nonstructural proteins are mainly involved in RNA replication. RNA replication is regulated leading to an asymmetric ratio of plus strand molecules to minus strand RNA molecules of 10:1. The genome RNAs assemble together with several copies of the viral capsid protein to form nucleocapsids that acquire a lipid bilayer envelope with virion glycoproteins by budding through intracellular membranes. Interestingly, recent studies also indicate a role of nonstructural proteins in infectious particle formation. Those as well as other studies describing functional analyses of yellow fever viral proteins have been performed using reverse genetics. An infectious cDNA clone has been described for the yellow fever virus vaccine strain 17D, which certainly is a great tool to gain further insights into the molecular biology of yellow fever virus.
Chapter 2
Title: Taxonomy and Evolution of the Flaviviridae
Ernest A. Gould and Tamara S. Gritsun
Abstract
Currently there are at least 73 characterized flaviviruses which on the basis of their phylogenetic relationships can be broadly divided into four groups. This number is increasing as more viruses are discovered and will undoubtedly continue to increase for some time yet. Some flaviviruses such as Yellow fever virus, Japanese encephalitis virus and Tick-borne encephalitis virus were first recognized because they caused major human epidemics involving high fatality rates. Others, such as dengue virus appear to have increased their capacity to cause severe disease as they have dispersed more widely throughout the tropics. In contrast others may not have been observed to produce overt disease but have been discovered either using conventional isolation and serological methods during the mid-twentieth century or more recently using improved methods such as RT-PCR sequencing for their recognition and identification. These new molecular methods have also revealed the occurrence of homologous recombination and the presence of flaviviral DNA in infected cells, the significance of which may not yet have been fully recognized. This review will focus on the taxonomy, evolution, emergence and dispersal of the flaviviruses taking into account recent new observations.
Chapter 3
Origin, Evolution and Function of Flavivirus RNA in Untranslated and Coding Regions: Implications for Virus Transmission
T. S. Gritsun, A. Tuplin and E. A. Gould
Abstract
In this review we analyse the research that specifically targets flavivirus RNA secondary structures. We focus mainly on data related to the 5' and 3' untranslated regions (5'UTR and 3'UTR) but the limited data relating to stable conserved secondary structures in the coding region of the flavivirus genome are also discussed. We propose that the 3'UTR, and possibly the open-reading frame, evolved through multiple duplication events of an RNA domain approximately 200 nucleotides in length, the remnants of which will be demonstrated in tick-borne flaviviruses. Subsequently, these repeat sequences and the associated RNA secondary structures may have evolved into stem-loop conformations with promoter and enhancer functions that impact on virus replication efficiency. The viral promoter probably folds as a complex transitional flexible RNA structure consisting of a number of transient stems and loops conserved between all flaviviruses. One of the transient forms of the promoter is formed due to the physical interaction between multiple complementary sequences in the 3'UTR, the 5'UTR and the coding region resulting in genome circularization. The folding of the 3'UTR, independently from the 5'UTR, revealed other transient promoter elements that might occur before or after circularization. These include a terminal 3' stable long hairpin (3'LSH) with an adjacent dumbbell-like structure DB1. The folding of the 5'UTR predicts the formation of a conserved terminal Y-shaped structure that is essential for virus infectivity and might contribute to the promoter function. The replication enhancer is located in the 3'UTR between the stop codon and the promoter. It contains repeated conserved sequences and secondary structures but it is more variable between different flaviviruses than the promoter. Although the enhancer function may not be essential for virus viability under experimental conditions in the laboratory, it might play a significant role in nature where the rate of virus replication could be critical for virus transmission and dissemination between vertebrates and invertebrates. The conserved RNA elements predicted in the coding region of the flavivirus genome might also function to accelerate virus replication in the environment thereby enhancing the likelihood of virus survival.
Chapter 4
Cis and Trans-acting Determinants of Flaviviridae Replication
Sven-Erik Behrens and Olaf Isken
Abstract
The past decade has seen a considerable boost in knowledge on the molecular determinants and mechanisms that orchestrate the intracellular replication of the genomic RNA of Flaviviridae members such as the flaviviruses Kunjin/West Nile and Yellow fever, the pestiviruses bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), and hepatitis C virus (HCV). This progress was particularly triggered by the availability of cDNA constructs that permit the instant in vitro transcription of full-length infectious viral RNA molecules and of sub-genomic replication competent RNAs, the so called "RNA replicons". Moreover, novel experimental strategies combined genetics studies of the viral RNAs with biochemical methods and assay systems and allowed (i) to characterize the functions of the viral proteins and to determine whether the proteins operate in cis or trans throughout the assembly of the viral replication complex, (ii) to define the role of RNA elements in the translation and replication process of the viral genome, and (iii) to identify and characterize the function of host-factors participating in the viral life cycle. Along these lines, a particularly important question that is yet unsolved with all positive-strand RNA viruses concerns how viral protein and RNA synthesis is coordinated. This review provides a synopsis on the current state of the art and proposes a model on the initial steps of the viral RNA replication pathway.
Chapter 5
A New Approach to Dengue Epidemiology. Sequential Infection and ADE Hypothesis: The Story of a Dogma
Xavier Deparis
Abstract
Since the 20th century, dengue fever has become one of the leading causes of morbidity and mortality in tropical areas. Dengue virus is a mosquito-borne virus transmitted by Aedes mosquitoes. Four distinct serological types of this virus exist. According to the World Health Organization, a person infected by one of the four dengue viruses goes through a spectrum of illness ranging from classical dengue fever, a self-limiting illness characterised by high temperature, headache, myalgia, arthralgia to a more severe form, dengue hemorrhagic fever. For over 30 years, the pathogenesis of dengue was attributed to the presence of enhancing antibodies that are acquired during a primary infection and lead to an increase of infected cells, thereby an increase in viremia, during secondary infections. This hypothesis is termed antibody-dependent enhancement (ADE). Although extensively studied, the role of ADE in the pathogenesis of dengue remains unverified. A lot of proofs exist demonstrating that we need to transcend the ADE theory in order to progress in dengue fever knowledge. A lot of facts demonstrate that dengue is logically close to yellow fever, and it is time to carry out a global study in several countries to assess the validity of the ADE theory.
Chapter 6
Functional Role of prM Glycoprotein in Dengue Virus Replication
Nopporn Sittisombut, Poonsook Keelapang and Prida Malasit
Abstract
Proteolytic cleavages of the prM protein during the replication of flaviviruses are intimately involved with the production and maturation of virion. Cleavage at the amino-terminus of prM by signal peptidase is coordinately regulated with the viral protease cleavage of anchored C protein to optimize the production of virions with nucleocapsid. Internal cleavage of prM on immature virion by furin allows rearrangement of the receptor-binding and viral fusion protein, E, enabling E to mediate envelope-membrane fusion upon exposure to low pH environment. Despite its importance in the generation of infectious virion, cleavage of prM is variable among different flaviviruses, partly due to the influence of charged residues within and proximal to the furin consensus sequence at the pr-M cleavage junction. Generation and characterization of pr-M junction chimeric and mutant viruses begin to unravel the structural basis for partial prM cleavage consistently observed in dengue viruses as well as the influence of cleavage on virus export. The remaining prM on extracellular dengue virion potentially serves as target for anti-prM antibodies, many of which exhibit infection-enhancing activity on dengue infection of Fc receptor bearing cells in vitro, but not strongly neutralizing effect. Several previous studies indicating the host-protective effect of anti-prM antibodies may reflect either their cross-reactivity with the E protein, or the contribution of remaining prM on post-binding step of the multiplication cycle.
Chapter 7
Diagnosis of Dengue Virus Infection
María G. Guzmán, Delfina Rosario and Gustavo Kourí
Abstract
The availability of a rapid, sensitive, specific and economical laboratory diagnostic test to confirm a dengue infection is of utmost importance in the management of Dengue Fever and Dengue Hemorrhagic Fever and in the prevention and control of outbreaks. In addition, travelers returning from endemic countries also constitute a diagnostic challenge to physicians. Virus detection through out virus isolation, antigen and genome detection allows a confirmatory serotype diagnosis. Antidengue IgM detection or high antidengue IgG titer in a serum sample or seroconversion in a paired of sera are the most routinely applied serological methods for dengue diagnosis.In spite of the advance of dengue diagnosis, some problems still warrant the timely development of new solutions. For instance, virus isolation is time consuming; PCR requires specific laboratory equipments and facilities as well as extensive evaluation of the different protocols under field conditions; IgM antibody detection requires proper timing and is confounded by false positive reactions and the long persistence of IgM antibodies, commercial kits still need to be critically evaluated, and the costs and availability of these kits and other reagents need to be addressed. In this chapter, an update of dengue diagnosis, its needs and challenges are discussed.
Chapter 8
Japanese Encephalitis Virus: Molecular Biology and Vaccine Development
Sang-Im Yun and Young-Min Lee
Abstract
Japanese encephalitis virus (JEV), the leading cause of viral encephalitis, frequently causes mortality and morbidity, especially in children and young adults in Asia. The recent geographic expansion of JEV activity has become a worldwide public health threat. JEV is a mosquito-borne flavivirus with a single-stranded, positive-sense RNA genome approximately 11,000 nucleotides in length. Recent development of an efficient reverse genetics system for JEV, using a genetically stable infectious cDNA as a bacterial artificial chromosome, is enabling advancement of direct molecular genetic studies previously hampered by the difficulty of manipulating the viral genome. Although considerable information has been learned about JEV biology from comparison with other closely related flaviviruses, much more needs to be elucidated and demonstrated experimentally. This review discusses the molecular aspects of common features of conserved sequence elements and viral gene products involved in viral replication, as compared to other flaviviruses. This will assist current strategies for development of a safer, more effective, and less expensive antiviral vaccine against this pathogen as well as closely related flaviviruses.
Chapter 9
Evolution, Epidemiology and Control of the Japanese Encephalitis Virus
Ma Shao-Ping and Makino Yoshihiro
Abstract
Research on the classification of the Japanese encephalitis (JE) virus in Japan has been related to the control of JE with vaccine. To develop a more suitable vaccine, epidemiological studies have been carried out in Japan. Until the mid-1980s, research to classify field isolates based on their antigenicity has been limited. The molecular epidemiological methods (finger printing and phylogenetic analyses) have drastically revealed that 1) JE viruses are composed of five geographically related but distinct genotypes, 2) new genotypes have occasionally been introduced into new geographical areas. Japan has successfully reduced the numbers of JE cases. The reduction of JE cases was mainly due to vaccination. Water control of rice fields also resulted in the reduction of the number of vector mosquitoes. Lastly, we discuss the recent vaccine developments (cell line derived JE vaccine and some new developing vaccines) and the antigenicity of genotype 1 strains recently introduced into Japan.
Chapter 10
Maturation and Assembly of Hepatitis C Virus Core Protein
Tetsuro Suzuki and Ryosuke Suzuki
Abstract
Hepatitis C virus (HCV) core protein, which is derived from the N-terminus of the viral polyprotein, forms the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains, compared to other HCV proteins. The N-terminal domain of the core protein is highly basic, while its C-terminus is hydrophobic. The core protein is primarily detected in the cytoplasm in associating with endoplasmic reticulum (ER), lipid droplets, and mitochondria. However, it has also been detected in the nucleus. In fact, a nuclear localization signal has been identified in the N-terminal region of the protein. Its C-terminal hydrophobic region is thought to act as a membrane anchor for the core protein and as a signal sequence for E1 protein. Nucleocapsid formation presumably involves interactions between the core protein and viral RNA and envelope protein(s), as well as self-interaction of the core protein. Interactions between the HCV core protein and specific regions of viral genomic RNA have been reported. In addition, the core protein has been observed to form homo-multimeric, as well as hetero-dimeric, complexes with E1 protein. These interactions might be important for nucleocapsid formation, and may activate the biological activity of various HCV structural proteins.
Chapter 11
Molecular Virology and Pathogenesis of Hepatitis C
Darius Moradpour and Hubert E. Blum
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Exciting advances have recently been made in the understanding of the molecular virology and pathogenesis of hepatitis C. These advances have translated into the identification of novel antiviral targets and the development of innovative therapeutic and preventive strategies, some of which are already in early phase clinical evaluation. Much work remains to be done with respect to the virion structure, the early and late steps of the HCV life cycle, the mechanism and regulation of RNA replication, and the pathogenesis of HCV-induced liver disease. However, given the current pace of HCV research, progress in these areas may be expected in the near future.
Chapter 12
Treatment of HCV-related Liver Diseases and Prevention of Hepatocellular Carcinoma
Kyuichi Tanikawa
Abstract
HCV-related liver diseases are characterized by persistent inflammation and the frequent development of hepatocellular carcinoma (HCC). These characteristics are due to immunological alterations caused by dysfunction of the dendritic cells and by oxidative stress in the hepatocytes. Immunological alterations brought about by persistent HCV infection and oxidative stress are among the most important risk factors for the development of HCC. Therefore, the most important clinical targets for the treatment of HCV-related liver diseases are the elimination of HCV from the body and the prevention of HCC. For these purposes Interferon treatment has been carried out with some success. Reduction of oxidative stress is also an important target in the treatment of HCV-related liver diseases and prevention of HCC.
Chapter 13
A New Perspective in The Pathophysiology Of Hepatitis C Virus (HCV) Infection: Interaction of the NS3 Protein of HCV With Serine-/Threonine-Specific Protein Kinases
Matthias Kalitzky, Holger Rohde and Peter Borowski
Abstract
Hepatitis C virus (HCV) causes aggressive infections of the liver with a high percentage of chronic courses. The pathophysiology of this finding has not been clarified on a molecular level. The present work describes a new aspect of the pathophysiology of this HCV related disease: the interference of viral antigens in intracellular signal transduction pathways. The analysis of the amino acid sequence of the product of the HCV genome revealed an arginine rich motif localized between the amino acids 1487 and 1500 of the HCV polyprotein. This sequence shows close similarity to arginine-rich motifs localized in the regulatory domains of the cAMP-dependent protein kinase A (PKA) and in the phospholipid/Ca2+-dependent protein kinase C (PKC). These motifs determine the recognition of the substrates and the kinetic activity of the protein kinases. A synthetic peptide with the amino acid sequence of the arginine rich motif of HCV inhibits the phosphorylation reaction mediated by the two enzymes by a competitive modus. Recombinant HCV proteins with the sequence of the arginine rich motif do not only block the catalytic activity of the protein kinases, but also inhibit further functions of these enzymes like their translocation between cell compartments and association with specific receptor proteins. Considering the important role of these protein kinases in cell growth, differentiation, carcinogenesis, and tumor promotion, it can be assumed that chronic impairment of the functions of these signal proteins by viral antigens can represent an essential part in the pathogenesis of HCV infection and in the carcinogenesis associated with HCV. The specific inhibition of interactions of these domains with cellular proteins can be used as a basis for the development of new therapeutic strategies.
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