Developments in Molecular Virology – serie

Fish lymphocystis disease virus (FLDV) has been tentatively classified as a separate genus of the iridovirus family. Iridoviruses were previously called icosahedral cytoplasmic deoxyriboviruses (ICDV). The Iridoviridae family contains four genera including lymphocystis disease virus (proposed name Lymphocystivirus, Willis, 1989 (1". FLDV is a causative agent of lymphocystis disease (LD) which frequently appears in Pleuronectidae (flatfish) such as Pleuronectes platessa (plaice), Platichtys flesus (flounder), Limanda limanda (dab), and Trigla gurnardus (gurnard). Fish lymphocystis disease is characterized by papilloma-like lesions, which can be induced experimentally in Lepomis macrochirus (bluegill) (2) and by subdermal injection of plaice and flounder (3). The mechanisms of this nonmalignant tumor induction are unknown. Since the discovery of LD in 1874 by Lowe (4), attempts have been made to isolate and propagate FLDV in vitro with limited success (5-7). As a first step towards understanding of the underlying mechanisms of this infectious disease the structure and properties of the causal virus must be eluciated.These basical molecular biological studies provide new facilities for investigation of virus host interactions which is necessary for understanding the molecular mechanisms of the viral pathogenesis. MATERIALS AND METHODS A total of 30 fish with LD lesions caught near the Doggerbank areas were analyzed individually, including 20 flounders, six dabs, and four plaice. Virions of FLDV from LD lesions of each species of fish were isolated, purified, and examined by electron microscopy as 205 described previously (8). Fig.

Biosynthesis of cellular and viral DNA and RNA has been a major topic in molecular biology and biochemistry. The studies by Arthur Kornberg and his colleagues on the in-vitro synthesis of DNA have opened new avenues to understanding the processes controlling the duplication of the genetic information encoded in the DNA and RNA of bacterial and mammalian cells. Viral nucleic acids are replicated in infected cells (bacterial, plant, and animal) by virus­ coded enzymes with or without the involvement of proteins and enzymes coded by the host cells. The ability of the virus to replicate its genome within a relatively short period in the infected cell makes it an excellent biological tool for studying the molecular events in nucleic acid replication. Indeed, the identification of a number of virus-coded proteins that participate in the biosynthesis of X174 and SV40 DNA has led to the construction of in-vitro systems for the study of nucleic acid biosynthesis. Similarly, studies on the replication of other phage, animal and plant viruses have provided an insight into the nucleic acid sequences from which DNA synthesis is initiated, as well as the proteins and enzymes that regulate the catalyse biosynthetic processes. Investi­ gation of the molecular processes involved in the replication of cellular and mitochondrial genomes has gained momentum from the rapid developments in the analyses of viral nucleic acid biosynthesis.

Venezuelan equine encephalitis (VEE) virus was first isolated in 1938 by Kubes and Rios (1) from the brain of a horse which died during an epizootic of a previously unrecognized disease in Venezuela. VEE-related viruses were subsequently isolated during t~e period of 1943-1963 in Venezuela, Colombia, Peru, Trinidad, Brazil, Surinam, Argentina, Panama, Mexico, and the United States (2) * Shope et ~. (3) fi rst defi ned the vi ru ses in the VEE comp 1 ex t-y showing serological relationships between classical VEE, ~lucambo, and Pixuna viruses. Young and Johnson (2) serologically characterized a variety of VEE isolates and proposed that the complex t>e divided into four subtypes (I, II, III, and IV). Viruses in subtype I were divided into five variants designated IA through IE. During 1069-1~71 a VEE epizootic-epidemic occurred in South America, Central America, and the United States involving a subtype lAB virus which caused high mortality among equines and human d i sea se (4). Venezuelan equine encephalitis viruses are alpha-togaviruses w~ic~ contain a positive strand rit>onucleic acid genome enclosed in an icosa~edral nucleocapsid.The virion has an envelope which contains blO glycoproteins: E2 of 5F,000 daltons (gp56) and E1 of ~O,OOO daltons (gp50) (5,6). Viral neutralization (N) and hemagglutiration (HA) sites have been placed on E2 by the use of monospecific rabtdt antisera and monoclonal antibodies specific for purified viral structural proteins (7-10). Only anti-E2 antisera neutralized virus infectivity or blocked virus hemagglutination.

Research on antiviral drugs and their mode of action in infected cells. in animals and in man. has led to a better understanding of the molecular pro­ cesses involved in virus replication. Screeninq of large numbers of natural and semisynthetic compounds resulted in the characterization of certain sub­ stances that had a limited efficiency as antiviral druqs. A few chemically synthesized compounds were also found to be effective as antiviral agents in the chemotherapy of human virus diseases. A major difficulty in the develop­ ment of effective antiviral agents has been the lack of selectivity. and toxicity for uninfected cells. of drugs that effectively inhibited virus replication in vitro. Further understanding of the molecular processes of virus replication in infected cells has resulted in the development of new antivirals directed at virus-coded enzymes or proteins. Recent studies on antivirals that are activated by the herpes simplex virus type l-coded thy­ midine kinase from a prod rug to an antiviral drug have opened new directions in the development of effective antiviral drugs. The present book deals with a number of antiviral drugs effective against herpes simplex viruses and provides some insight into the molecular aspects of virus replication. It also throws light on the new approaches to the development of antiviral drugs. The molecular basis of the antiviral activity of new and known drugs and their possible use in chemotherapy of viral disease are presented in this book.

The development of recombinant DNA technology has made a marked impact on molecular virology. The cleavage of viral DNA genomes with restriction enzymes and the cloning of such DNA fragments in bacterial p1asmids has led to the amplification of selected viral DNA fragments for sequencing and gene expression. RNA virus genomes which can be transcribed to their cDNA form were also cloned in bacterial p1asmids, facilitating the study of RNA virus genes. With the elucidation in recent years of the promoter sequence of various viral genes and the expression of these genes in bacteria or yeast, the understanding of many viral gene functions has made great progress. Cloning and expression of viral genes in mammalian cells was made possible by the construction of shuttle plasmid vectors which carry the origins of DNA replication from bacteria and/or mammalian viruses. The expression of viral genes in bacteria, yeast and eukaryotic cells gives reason to hope that it will be possible to produce viral antigens in large quantities for use as human or animal vaccines. The present volume attempts to capture for the reader some of the high­ lights of recombinant DNA research in the field of animal and plant viruses.

Since the discovery of Australia antigen and its association with type B hepatitis, molecular characterization of the components making up hepatitis B virus (RBV) have been pursued with worldwide interest. Over the past two decades, such characterization has led to the development of sensitive assays to screen and exclude contaminated units from blood banks and has recently resulted in the licensing of several RBV vaccines. That more than 200 million people worldwide are chronically infected with RBV, and that they are at a high risk for the development of chronic hepatitis and hepatocellular carcinoma, still represent formidable problems in our understanding of host-virus relationships on the molecular level. In the absence of a suitable tissue culture system, and with a very limited host range of infection, characterization of RBV on the molecular level has made remarkable progress recently with the advent of genome cloning, sequencing and expression of individual virus genes by recombinant DNA technology. The presence of hepatitis B-like viruses in an expanding number of animal hosts, and the possibility of virus replication in cells other than hepatocytes, provide great promise that future work will elucidate the molecular mechanisms operative in the various outcomes of RBV infection.

The nucleotide sequence of the gene from which messenger RNA mole­ cules are transcribed is in a form that can be translated by cellular ribosomes into the amino acid sequence of a particular polypeptide, the product of the gene. The discovery of messenger RNA more than twenty years ago led to a series of studies on its organization and function in cells in the presence of infecting viruses. This volume is devoted to current studies in the field of cellular and viral messenger RNA. The studies presented provide an insight into molecular and genetic aspects of messenger RNA. Special attention was paid by the authors to the molecular organization of mRNA species, to the processing of mRNA molecules, and to the different strategies employed by DNA and RNA viruses in the synthesis of their mRNA. The ability of a virus to take over the protein-synthesizing mechanisms of an infected cell depends on its ability to produce mRNA molecules which can affect the host mRNA or utilize cellular components more efficiently. The differences between, and similarities of, the strategies of mRNA synthesis devised by various DNA and RNA viruses are described herein. This book should be of interest to all students of cellular and viral genes and scientists in the field. It is suitable as a textbook for workshops and courses on mRNA. I wish to thank the authors for their fine contributions and for their interest.

Although adenoviruses have been established for quite some time as one of the most pliable tools to study the molecular biology of mammalian cells, rapid progress continues to be made with this virus system. The adenoviral genome introduced into the nucleus of cells as a Trojan Horse, as it were, is now facili- tating investigators to uncover details of cellular mechanisms. In this volume twelve chapters have been collected that summarize important current research on different mechanisms in adenovirus- infected and -transformed cells. It has become increasingly apparent that some of these mechanisms do not only pertain to highly specialized strategies of the viral genome and its expres- sion or replication, but may simultaneously shed light on events indigenous to the cell. "Adenovirus DNA: The Viral Genome and its Expression" high- lights the first publication of the entire annotated sequence of 35. 937 nucleotide pairs of adenovirus type 2 DNA by the Cold Spring Harbor and Uppsala groups (chapter 1). Goran Akusjarvi, Ulf Pettersson (Uppsala University) and Rich Roberts (Cold Spring Harbor) present a survey on the structure and function of the adenovirus-2 genome (chapter 2).A considerable amount of in- formation has been collected on the biochemical mechanisms in- vol ved in adenov irus DNA repl ication in human cells. Al though highly specialized in the way of initiating DNA replication, adenov irus DNA repl ication represents probably one of the best studied systems in work on eukaryotic DNA replication.

It is almost twenty years since the first DNA tumor virus meeting was held at Cold Spring Harbor. At this meeting studies on three tumor viruses were discussed: the papovaviruses, the adenoviruses and the herpesviruses. The present series Developments in Molecular Virology chose to reverse this sequence by first publishing books on the herpesviruses, followed by adenoviruses, and only now the papo­ vaviruses. All the DNA tumor viruses gained their original reputation by serving as model systems in animal cells for studying gene expression and gene regulation, but SV40 and polyoma have been the jewel in the crown in these studies, as A phage was for the study of prokaryotes. SV40 was the first DNA tumor virus to be completely sequenced that enabled the definition of the cis controlling elements in DNA replication and transcription. I am continuously fascinated by the organization of the SV40 and polyoma genomes. Although they contain about 5000 bp that encode for only 6 to 7 proteins, the mechanisms which regulated their gene expression are varied and include almost any other type of gene regulation found today to regulate eukaryotic genes. Just to mention two: (i) the early promoter is a classical promoter that contains the TAT A, CAAT and enhancer elements, while the late promoter is devoid of these elements, and (ii) the mRNA can be structurally and functionally monocistronic or dicistronic. This hints at the versatility in the control of gene expression at the transcriptional and translational levels.

Fish lymphocystis disease virus (FLDV) has been tentatively classified as a separate genus of the iridovirus family. Iridoviruses were previously called icosahedral cytoplasmic deoxyriboviruses (ICDV). The Iridoviridae family contains four genera including lymphocystis disease virus (proposed name Lymphocystivirus, Willis, 1989 (1". FLDV is a causative agent of lymphocystis disease (LD) which frequently appears in Pleuronectidae (flatfish) such as Pleuronectes platessa (plaice), Platichtys flesus (flounder), Limanda limanda (dab), and Trigla gurnardus (gurnard). Fish lymphocystis disease is characterized by papilloma-like lesions, which can be induced experimentally in Lepomis macrochirus (bluegill) (2) and by subdermal injection of plaice and flounder (3). The mechanisms of this nonmalignant tumor induction are unknown. Since the discovery of LD in 1874 by Lowe (4), attempts have been made to isolate and propagate FLDV in vitro with limited success (5-7). As a first step towards understanding of the underlying mechanisms of this infectious disease the structure and properties of the causal virus must be eluciated.These basical molecular biological studies provide new facilities for investigation of virus host interactions which is necessary for understanding the molecular mechanisms of the viral pathogenesis. MATERIALS AND METHODS A total of 30 fish with LD lesions caught near the Doggerbank areas were analyzed individually, including 20 flounders, six dabs, and four plaice. Virions of FLDV from LD lesions of each species of fish were isolated, purified, and examined by electron microscopy as 205 described previously (8). Fig.

It is almost twenty years since the first DNA tumor virus meeting was held at Cold Spring Harbor. At this meeting studies on three tumor viruses were discussed: the papovaviruses, the adenoviruses and the herpesviruses. The present series Developments in Molecular Virology chose to reverse this sequence by first publishing books on the herpesviruses, followed by adenoviruses, and only now the papo­ vaviruses. All the DNA tumor viruses gained their original reputation by serving as model systems in animal cells for studying gene expression and gene regulation, but SV40 and polyoma have been the jewel in the crown in these studies, as A phage was for the study of prokaryotes. SV40 was the first DNA tumor virus to be completely sequenced that enabled the definition of the cis controlling elements in DNA replication and transcription. I am continuously fascinated by the organization of the SV40 and polyoma genomes. Although they contain about 5000 bp that encode for only 6 to 7 proteins, the mechanisms which regulated their gene expression are varied and include almost any other type of gene regulation found today to regulate eukaryotic genes. Just to mention two: (i) the early promoter is a classical promoter that contains the TAT A, CAAT and enhancer elements, while the late promoter is devoid of these elements, and (ii) the mRNA can be structurally and functionally monocistronic or dicistronic. This hints at the versatility in the control of gene expression at the transcriptional and translational levels.

Although adenoviruses have been established for quite some time as one of the most pliable tools to study the molecular biology of mammalian cells, rapid progress continues to be made with this virus system. The adenoviral genome introduced into the nucleus of cells as a Trojan Horse, as it were, is now facili- tating investigators to uncover details of cellular mechanisms. In this volume twelve chapters have been collected that summarize important current research on different mechanisms in adenovirus- infected and -transformed cells. It has become increasingly apparent that some of these mechanisms do not only pertain to highly specialized strategies of the viral genome and its expres- sion or replication, but may simultaneously shed light on events indigenous to the cell. "Adenovirus DNA: The Viral Genome and its Expression" high- lights the first publication of the entire annotated sequence of 35. 937 nucleotide pairs of adenovirus type 2 DNA by the Cold Spring Harbor and Uppsala groups (chapter 1). Goran Akusjarvi, Ulf Pettersson (Uppsala University) and Rich Roberts (Cold Spring Harbor) present a survey on the structure and function of the adenovirus-2 genome (chapter 2).A considerable amount of in- formation has been collected on the biochemical mechanisms in- vol ved in adenov irus DNA repl ication in human cells. Al though highly specialized in the way of initiating DNA replication, adenov irus DNA repl ication represents probably one of the best studied systems in work on eukaryotic DNA replication.

The development of recombinant DNA technology has made a marked impact on molecular virology. The cleavage of viral DNA genomes with restriction enzymes and the cloning of such DNA fragments in bacterial p1asmids has led to the amplification of selected viral DNA fragments for sequencing and gene expression. RNA virus genomes which can be transcribed to their cDNA form were also cloned in bacterial p1asmids, facilitating the study of RNA virus genes. With the elucidation in recent years of the promoter sequence of various viral genes and the expression of these genes in bacteria or yeast, the understanding of many viral gene functions has made great progress. Cloning and expression of viral genes in mammalian cells was made possible by the construction of shuttle plasmid vectors which carry the origins of DNA replication from bacteria and/or mammalian viruses. The expression of viral genes in bacteria, yeast and eukaryotic cells gives reason to hope that it will be possible to produce viral antigens in large quantities for use as human or animal vaccines. The present volume attempts to capture for the reader some of the high­ lights of recombinant DNA research in the field of animal and plant viruses.

Since the discovery of Australia antigen and its association with type B hepatitis, molecular characterization of the components making up hepatitis B virus (RBV) have been pursued with worldwide interest. Over the past two decades, such characterization has led to the development of sensitive assays to screen and exclude contaminated units from blood banks and has recently resulted in the licensing of several RBV vaccines. That more than 200 million people worldwide are chronically infected with RBV, and that they are at a high risk for the development of chronic hepatitis and hepatocellular carcinoma, still represent formidable problems in our understanding of host-virus relationships on the molecular level. In the absence of a suitable tissue culture system, and with a very limited host range of infection, characterization of RBV on the molecular level has made remarkable progress recently with the advent of genome cloning, sequencing and expression of individual virus genes by recombinant DNA technology. The presence of hepatitis B-like viruses in an expanding number of animal hosts, and the possibility of virus replication in cells other than hepatocytes, provide great promise that future work will elucidate the molecular mechanisms operative in the various outcomes of RBV infection.

The nucleotide sequence of the gene from which messenger RNA mole­ cules are transcribed is in a form that can be translated by cellular ribosomes into the amino acid sequence of a particular polypeptide, the product of the gene. The discovery of messenger RNA more than twenty years ago led to a series of studies on its organization and function in cells in the presence of infecting viruses. This volume is devoted to current studies in the field of cellular and viral messenger RNA. The studies presented provide an insight into molecular and genetic aspects of messenger RNA. Special attention was paid by the authors to the molecular organization of mRNA species, to the processing of mRNA molecules, and to the different strategies employed by DNA and RNA viruses in the synthesis of their mRNA. The ability of a virus to take over the protein-synthesizing mechanisms of an infected cell depends on its ability to produce mRNA molecules which can affect the host mRNA or utilize cellular components more efficiently. The differences between, and similarities of, the strategies of mRNA synthesis devised by various DNA and RNA viruses are described herein. This book should be of interest to all students of cellular and viral genes and scientists in the field. It is suitable as a textbook for workshops and courses on mRNA. I wish to thank the authors for their fine contributions and for their interest.

Research on antiviral drugs and their mode of action in infected cells. in animals and in man. has led to a better understanding of the molecular pro­ cesses involved in virus replication. Screeninq of large numbers of natural and semisynthetic compounds resulted in the characterization of certain sub­ stances that had a limited efficiency as antiviral druqs. A few chemically synthesized compounds were also found to be effective as antiviral agents in the chemotherapy of human virus diseases. A major difficulty in the develop­ ment of effective antiviral agents has been the lack of selectivity. and toxicity for uninfected cells. of drugs that effectively inhibited virus replication in vitro. Further understanding of the molecular processes of virus replication in infected cells has resulted in the development of new antivirals directed at virus-coded enzymes or proteins. Recent studies on antivirals that are activated by the herpes simplex virus type l-coded thy­ midine kinase from a prod rug to an antiviral drug have opened new directions in the development of effective antiviral drugs. The present book deals with a number of antiviral drugs effective against herpes simplex viruses and provides some insight into the molecular aspects of virus replication. It also throws light on the new approaches to the development of antiviral drugs. The molecular basis of the antiviral activity of new and known drugs and their possible use in chemotherapy of viral disease are presented in this book.

Biosynthesis of cellular and viral DNA and RNA has been a major topic in molecular biology and biochemistry. The studies by Arthur Kornberg and his colleagues on the in-vitro synthesis of DNA have opened new avenues to understanding the processes controlling the duplication of the genetic information encoded in the DNA and RNA of bacterial and mammalian cells. Viral nucleic acids are replicated in infected cells (bacterial, plant, and animal) by virus­ coded enzymes with or without the involvement of proteins and enzymes coded by the host cells. The ability of the virus to replicate its genome within a relatively short period in the infected cell makes it an excellent biological tool for studying the molecular events in nucleic acid replication. Indeed, the identification of a number of virus-coded proteins that participate in the biosynthesis of X174 and SV40 DNA has led to the construction of in-vitro systems for the study of nucleic acid biosynthesis. Similarly, studies on the replication of other phage, animal and plant viruses have provided an insight into the nucleic acid sequences from which DNA synthesis is initiated, as well as the proteins and enzymes that regulate the catalyse biosynthetic processes. Investi­ gation of the molecular processes involved in the replication of cellular and mitochondrial genomes has gained momentum from the rapid developments in the analyses of viral nucleic acid biosynthesis.

Venezuelan equine encephalitis (VEE) virus was first isolated in 1938 by Kubes and Rios (1) from the brain of a horse which died during an epizootic of a previously unrecognized disease in Venezuela. VEE-related viruses were subsequently isolated during t~e period of 1943-1963 in Venezuela, Colombia, Peru, Trinidad, Brazil, Surinam, Argentina, Panama, Mexico, and the United States (2) * Shope et ~. (3) fi rst defi ned the vi ru ses in the VEE comp 1 ex t-y showing serological relationships between classical VEE, ~lucambo, and Pixuna viruses. Young and Johnson (2) serologically characterized a variety of VEE isolates and proposed that the complex t>e divided into four subtypes (I, II, III, and IV). Viruses in subtype I were divided into five variants designated IA through IE. During 1069-1~71 a VEE epizootic-epidemic occurred in South America, Central America, and the United States involving a subtype lAB virus which caused high mortality among equines and human d i sea se (4). Venezuelan equine encephalitis viruses are alpha-togaviruses w~ic~ contain a positive strand rit>onucleic acid genome enclosed in an icosa~edral nucleocapsid.The virion has an envelope which contains blO glycoproteins: E2 of 5F,000 daltons (gp56) and E1 of ~O,OOO daltons (gp50) (5,6). Viral neutralization (N) and hemagglutiration (HA) sites have been placed on E2 by the use of monospecific rabtdt antisera and monoclonal antibodies specific for purified viral structural proteins (7-10). Only anti-E2 antisera neutralized virus infectivity or blocked virus hemagglutination.

Herpesviruses, classified in the family Herpesviridae, are important human and animal pathogens that can cause primary, latent or recurrent infections and even cancer. The major interest in research on herpesviruses today focuses on understanding the organization of the DNA genome, as well as on characterizing the viral genes in regard to their control and function. Modern techniques have allowed the viral DNA to become a molecular tool in the study of gene function, since it is now possible to implant the DNA into eukaryotic cells. This book contains original studies on the structure and organization of the DNA of human and animal herpes­ viruses. The various chapters acquaint the reader with the organization of the viral DNA, the mRNA transcripts, the replicative intermediates of the viral DNA, defective DNA genomes and their mode of synthesis, analyses of the viral DNA sequences in transformed cells, and the relationship between the presence of viral DNA fragments in the cancer cells and the transformed state of the cells.