Eckard Wimmer - US grants

We propose to continue and greatly extend our studies on the molecular biology of picornaviruses, particularly on poliovirus. We shall study aspects of viral protein synthesis (initiation and processing of polypeptides) and the intriguing problem of viral RNA replication. We will use recombinant DNA technology to define gene function (in vitro recombination, site specific mutagenesis) and we plan to characterize the surface properties of the virion (neutralizing epitopes, virus-cell attachment sites). Our studies may lead to the development of new vaccines and drugs directed against picornavirus infection.

We propose to continue and extend our studies on molecular events leading to poliovirus replication and our analysis of neutralization antigenic sites of the virus particle. Specifically, we propose to analyze the structure and function(s) of the 5' non- coding region of poliovirus RNA, to determine processes of protein modification and of proteolytic cleavage of the poliovirus polyprotein, to investigate the structure and substrate recognition of the two virus-encoded proteinases, and to analyze components of viral RNA replication and the mechanism of initiation of viral RNA synthesis. Moreover, we will alter the surface structure of the poliovirion to achieve hybrid virus expressing foreign antigenic determinants in addition to those of poliovirus Type 1 (Mahoney). Our studies may lead to the development of new vaccines and of novel drugs directed against picornavirus infection.

This proposal deals with translational control of gene expression in mammalian cells which takes a unique position in cellular metabolism. We use a viral system (the picornaviruses) to study the properties and function of two cellular factors, P57 and "initiation correcting factor" (ICF), that we have described previously. Factor p57 has been shown to be essential in the function of IRES (internal ribosomal entry site), a structural element in the 5'non-translation region (5'NTR) of picornavirus mRNA that confers cap-independence of translation. The ICF corrects false initiation of translation of poliovirus RNA in rabbit reticulocytes lysates. The role of the factors in cellular translation is not known. We shall isolate the factors, clone the corresponding cDNAs, determine their sequence, and prepare factor-specific immunological probes. We will purify large amounts by over-expression in suitable cells, and study their biochemical properties, and the effect of possible post-translational modifications with respect to their interaction with picornavirus 5'NTRs. Of particular interest is whether the factors occur unmodified in normal quantities in human neuronal cells, and whether the interaction of either factor with RNA of attenuated polioviruses is modified. These studies will contribute to the molecular understanding of the attenuation phenotype of poliovirus. They will also characterize cellular RNA binding proteins, a class of factors whose interaction with RNA and whose role in cellular metabolism is of central importance yet poorly understood.

DESCRIPTION (provided by applicant): It is the objective of the work proposed in this grant application to enhance our understanding of the mechanisms by which poliovirus (PV), an enterovirus belonging to the Picornaviridae, encapsidates its genome. This virus family includes a large number of human and animal pathogens that cause more than 6 billion human infections worldwide each year. These infections lead to a variety of diseases ranging from the mild (common cold) to the serious (poliomyelitis). In spite of research for many years the details of most steps in the life cycle of PV remain unknown. However, the proliferation of PV remains an important medical issue because epidemic PV infections are expected to occur even after the circulation of wt PV is interrupted globally. This proposal can be divided into 3 parts. The first aim deals with the development of new genetic tools to study the genetics of encapsidation of chimeras constructed from PV and the closely related C-cluster coxsackie A viruses. We expect that the morphogenesis phenotypes of the chimeric viruses will be very useful in analyzing the interaction of capsid and nonstructural proteins during encapsidation. The second aim of the proposal deals with two specific inhibitors of enterovirus encapsidation, hydantoin and L-buthionine-sulfoximine (BSO), whose effect is expected to block encapsidation at different stages. In this study PV, CAVs and PV/C-CAV chimeras will be used to identify and analyze escape mutants in proteins involved in encapsidation. In our third aim we propose to analyze the role in morphogenesis of non-structural proteins 2CATPase, 3CDpro, VPg, and, and search for an elusive RNA encapsidation signal. We plan to use both genetic and biochemical studies to analyze the role of nonstructural proteins 2CATPase and 3CDpro in encapsidation. The role of VPg will be tested by a large-scale scan of hundreds of VPg mutants with the aim of finding replication positive but encapsidation negative mutants. Finally, we will use a novel strategy (codon-pair optimization) to scan the PV RNA for an elusive encapsidation signal. Since encapsidation is a uniquely viral process an understanding of its mechanism will aid the development of antiviral drugs that target this particular step in the enteroviral life cycle. The 2CATPase and 3CDpro proteins are highly conserved among enteroviruses hence they provide an excellent target for drug development to treat multiple enteroviral diseases. It is believed that these studies and results will be of interest not only to those investigators who study the encapsidation of other enteroviruses but also to those who are interested in picornaviruses or RNA viruses in general. PUBLIC HEALTH RELEVANCE: Picornaviridae cause about 6 billion human infections a year worldwide, some leading to serious diseases. Poliovirus, a member of the enterovirus genus, is the prototype of this virus family. The project proposed in this application deals with the mechanism of encapsidation of enteroviruses, particularly of poliovirus and of the closely related c-cluster enteroviruses. Our understanding of the mechanism of encapsidation is expected to facilitate the development of new drugs to treat enteroviral infections.

In this new application Dr. Wimmer proposes to examine a variety of different picornaviruses in the C cluster of coxsackievirus (C-CAV), which is the group that are defined on the basis of sequence similarities as containing poliovirus, and various coxsackieviruses, including CAV21 and CAV24. The overall objective of the proposal is to gain more information about those viruses, and also to develop new model systems from some of them. In these studies they will use their extensive knowledge and studies of poliovirus systems as a model for proposing the systems to be developed for the viruses to be studied here. There are a variety of different experiments proposed, and several different viruses will be examined in various parts of the proposed studies. During the course of the studies the investigators will make selections among the viruses in order to decide on the virus (es) to be studied in more detail. There are 4 specific aims. The first is to perform some very basic virological studies of the C-CAVs--but mainly on CAV21--to determine which of them can grow well in tissue culture, and then to study the growth characteristics and other general properties of the virion. Other studies will be of the antigenic properties of the virion, including the preparation and mapping of the neutralizing epitopes of the virus. The second specific aim is to define the genomic properties of the C-CAVs, including the preparation of full-length clones and infectious cDNAs of several of the viruses, in particular the Coxsackie A24 virus (CAV24), and the CAV24v that is a variant the causes hemorrhagic conjunctivitis. The sequences will also be used in studies of the evolutionary relationships between the various C-CAVs. Other studies would include the cell free de novo synthesis of CAV21, and various studies involving recombining genomic segments among the different C-CAVs in order to map their biological differences. The 3rd specific aim is to crystallize the capsid of one or more of those viruses, and then determine the structures of the virion by X-ray crystallography, in collaborative studies with Michael Rossmann and Richard Kuhn at Purdue University. The 4th specific goal is to define the interactions between the capsid and the receptors of those viruses, including ICAM-1 and DAF with CAV21a, and the relationships between CAV24 and CAV24v.

DESCRIPTION (provided by applicant): C-Cluster enteroviruses are a group of highly infectious igents within Picornav7 dae, genus Enterovirus, that are characterized by closely related genotypes but vastly different pathogenic properties (poliomyelitis vs. respiratory disease). They can be sub-divided into three poliovirus (PV) and 11 C-cluster coxsackie A virus (C-CAy) species that differ principally in their use of the cellular receptor (hPVR and ICAM-1, respectively). The clustering of these viruses is based on very limited knowledge regarding genotypes. The structure at the atomic level of C-CAV virions is unknown making it impossible to relate surface function to structure and, thus, to the evolution of virion/receptor interaction. We propose to analyze the relationship between C-cluster enteroviruses, aiming at elucidating the evolutionary pathway by which these viruses chose to become coxsackie or polioviruses. This will include the determination of genotypes and molecular biology of several C-CAVs, the elucidation of the crystal structure of a C-CAy virion and its antigenic properties, and the analysis of receptor/virion interaction. The relationship between poliovirus and its human host has dramatically changed during the twentieth century. New measures of hygiene practiced for the first time in industrialized countries at the beginning of last century led, paradoxically, to devastating poliomyelitis epidemics that, in turn, prompted the development of effective vaccines. Indeed, efforts are underway to eradicate poliovirus globally. We have entertained the possibility that in a world free of poliovirus and anti-polio antibodies some C-CA Vs may switch receptors (ICAM-l to CD155) to evolve as novel polioviruses. To assess this scenario we propose to establish a model system to test possibilities of speciation of C-cluster enteroviruses.

EUROPIC meetings are dedicated to review, every two years, progress and future directions of research related to Picornaviridae, one of the largest families of human and animal pathogenic viruses. So far, eleven previous meetings have been organized by the European Study Group on Molecular Biology of Picornaviruses, and they have all been held in (nine) European countries. Although this international meeting derived its name from the venue and the continent of origin of the majority of the attendees, the meeting has included Americans from the start and Americans now make up a sizeable fraction of attendees (>21% of the participants at EUROPIC 2000 in Italy. For this reason, the Americans were "nominated" during the last EUROPIC for the first time to organize EUROPIC/america 2002 in the US. A group of five active American picornavirologists volunteered to organize the first American picornaviruses meeting, to be held on Cape Cod at the SeaCrest Resort and Conference Center in Falmouth, MA, on May 14-19, 2002. The meetings have been traditionally attended by scientists from universities, research centers and industry, by postdoctoral fellows and graduate students Indeed, they have been characterized by a very large percentage of young investigators (particularly graduate students), a virtue that is highly educational and will be maintained. It has been estimated that picornaviruses cause >6 billion human infections/yr. and although most infections are self-limiting, a very large proportion cause serious if not fatal human disease. Moreover, new dangerous virus strains are emerging (see the recent outbreak of a new strain of enterovirus 71 in Taiwan). In addition, picornaviruses are the cause of serious animal diseases of which foot- and mouth disease virus (FMDW) may be cited as example. Apart from topics of epidemiology, mechanisms of pathogenesis, structure, and replication, it is planned to organize two workshops within the program that will cover topics of greatest current interest. The first will be dedicated to the cause of the foot-and-mouth disease epizootic ravaging England at the present time, its impact on society, and possibilities of prevention. The over will discuss the difficulties (circulation of vaccine strains, recent outbreak of poliomyelitis in the Western Hemisphere) that have emerged during the current, historic effect to globally eradicate poliovirus.

DESCRIPTION (provided by applicant): An Internal Ribosomal Entry Site (IRES) is a genetic element found in the genome of certain eukaryotic plus strand RNA viruses and cellular mRNAs. IRESs direct attachment of the 30S ribosomal subunit to a site downstream of the commonly used 5'terminal cap. IRES elements are large segments of RNA of different sequence that are highly structured. Neither the precise structure of these elements nor the precise mode of function is known. In this application, we propose to study the structure of the IRES of hepatitis C virus with respect structure and function. Furthermore, we will attempt to decipher the role of RNA binding proteins in the function of picornavirus and HCV IRES function. We have previously observed that an exchange of the poliovirus IRES element with that of rhinovirus type 2 results in a chimera, expressing an attenuated neurovirulence phenotype. We will investigate the molecular reason for this attenuation. Finally, we will determine whether we can exchange viral IRESs also IRESs of cellular mRNA.

Gene Design for Vaccines and Therapeutic Phages
PI: Steven Skiena

Vaccines and antibiotics have proven to be tremendously important agents against viral and bacterial infections, respectively. However, current vaccines are ineffective against certain viruses (e.g. rhinovirus) and potentially dangerous against others (e.g. smallpox). Further, the evolution of antibiotic-resistant strains of bacteria has become a critical problem in treating diseases such as tuberculosis.

We propose to exploit the redundancy inherent in the genetic code to create both safer and more effective vaccines and an improved class of anti-bacteriological agents. We apply design and synthesis techniques to replace viral genes so they code for identical proteins as in the wild type virus, but in different ways. For the first application, we seek to weaken viral strains by introducing mutations that alter translational efficiency and RNA secondary structure without affecting protein coding so as to create better vaccine candidates. For the second application, we seek to strengthen bacteriophages (viruses which attack bacteria) by eliminating important restriction sites so as to improve their ability to combat pathogenic infections.

The PI (Skiena) is a computer scientist who has worked extensively on theoretical sequence design problems which seek optimized DNA sequences which code for a particular protein while simultaneously accomplishing some other objective. The co-PI (Wimmer) is a microbiologist who attracted worldwide attention in July 2002 by synthesizing poliovirus completely from off-to-shelf components. But once you can synthesize an existing genome from scratch, you can do the same for new and better designs as well. We will collaborate on a series of computational and wet lab experiments using poliovirus to test and develop this vision.

Broader impacts of the proposed research include benefits to society through developing new types of medical therapeutics, and its impact on interdisciplinary bioinformatics education.

DESCRIPTION (provided by applicant): Following ingestion and entry into the gastrointestinal (Gl) tract, poliovirus, a human neurotropic enterovirus of Picornaviridae, may replicate in gastrointestinal-associated lymphatic tissues (GALT) and, subsequently, spread to the systemic circulation and to the CNS. Numerous attempts to study oral poliovirus infection in transgenic mice have so far failed. A focus of this application is to construct a novel transgenic mouse model for oral poliovirus infection that can be used to determine the site(s) of poliovirus proliferation in the Gl tract. A second focus of this application is to study mechanism(s) by which poliovirus is migrating in polarized tissue culture cells, in cells of neuronal origin, and in axons. Experiments have been developed based on our hypothesis that an interaction between the poliovirus receptor CD155 and Tctex-1, a cargo binding polypeptide of the dynein motor complex, is responsible for movement of poliovirus along microtubules.

[unreadable] DESCRIPTION (provided by applicant): Using modern methods for synthesizing long DNAs, and using computer algorithms, we have recently synthesized several totally novel variants of polio virus in vitro from oligonucleotides, and we have converted these nucleic acids into infectious virus. These viruses preserve the exact protein coding capacity of wild- type polio, but use synonymous codons in various ways to target translation. Two of the viruses were designed to have poor codon bias, or poor codon pair bias, respectively, and both of these viruses were inviable (i.e., cannot form plaques on cultured cells). Here, we seek to understand exactly why the altered viruses are attenuated; to find ways of generating still other, novel polio viruses attenuated to predictable extents; and to extend this approach of synthesizing predictably-attenuated viruses to other classes of viruses. The most important longer term implication of these studies is that the predictable synthesis of attenuated virus should provide a rapid, safe, inexpensive, general and reliable method of creating the raw material for viral vaccines. In principle, vaccines could be created quickly even for very poorly characterized viruses, as long as a nucleic acid sequence is available. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Vaccination has been humankind's main most robust defense against viral disease. We describe an entirely novel and rapid method to generate anti-virus vaccine candidates that might prove applicable to most if not all human pathogenic viral systems. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We plan to explore changes of the specific sequence space of genomes of arboviruses that have evolved to replicate efficiently in cells of two taxa (mammals and insects) at two different temperatures (27 and 37 C) and with a delicately balanced codon pair bias (CPB) accommodating CPB differences in mammals and insects. We have chosen for our studies dengue virus (DENV) that is the leading cause of arthropod-borne human diseases in the world. This is a truly multidisciplinary project that brings together investigators from a leading laboratory studying dengue virus pathogenesis and molecular biology (Berkeley, CA), from an Arbovirus Laboratory (Albany, NY) studying the biology of insect borne viruses, from a Department of Computer Sciences (University of Miami) specializing in encodings of biological system, and from a Department of Molecular Genetics and Microbiology (Stony Brook University, NY) known for work on plus stranded viruses. With the aid of computers and specifically tailored algorithms we will recode the genome of DENV such that it will contain large segments (encoding either the proteins E, NS3, or NS5) of codon pairs dis-favored in mammals but normal in insects. We predict that these genomes will encounter severe restrictions of replication in mammalian cells, but will replicate in insects cells with wild type kinetics. To avoid the possibility that the recoded viruses could be a biohazard for the environment (via Ae. aegypti, their natural vectors), the recoded viruses will be tested in mosquitoes for replication phenotypes. The recoded DENV genomes, in turn, will also be tested for their virulence and attenuation in experimental animals (mice and monkeys). Since the basic strategy allows us tailoring the virulence of the agent by specifically down-regulating the expression of virus- encoded proteins, our project offers a variety of possibilities to study replication phenotypes of DENV tissue culture cells and experimental animals. At the same time it may yield candidate strains that may be suitable for further development as vaccines. Vaccine development, however, is not the primary goal pursued in this application.