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Background: Synergy exists between DNA and RNA viruses. It was found that the Human Immunodeficiency Viruses (HIV-1) are RNA viruses at the origin of Acquired Immune Deficiency Syndrome (AIDS). The DNA recurrent herpes diseases are associated to AIDS virus at the origin of Sub-Saharan cancer AIDS pandemic. Objective: It is speculated that a varicella virus (HHV-3) immune defect could originate HSV- 1/HSV-2 recurrent herpes diseases that can be cured by varicella vaccine (2012). Methods: At a Symposium held in Kampala, Uganda (1962), impressive Sub-Saharan cancer epidemics: Hodgkin lymphomas and Kaposi sarcomas have been reported since the onset of the 20th century and remained unexplained. Over one thousand publications related to these cancer epidemics were presented. For millenniums, Bantu populations have been living in tropical forests close to chimpanzees infected by Simian Immune Deficiency viruses (SIV). SIV became Human Immune Deficiency viruses (HIV-1). AIDS is a zoonosis. Results: The DNA and RNA viruses, herpes with HIV-1 viruses, are correlated to Sub- Saharan AIDS infections. They induce an extensive immune deficiency with other herpes viruses such as HHV-4 and HHV-8, which are linked to lymphomas and Kaposi sarcomas. It is postulated that a primary HHV-3 immune weakness could be linked to herpes partnership with AIDS pandemic. Conclusion: The Oka, anti-HHV-3, varicella vaccine is able to cure HSV1/HSV2 recurrent herpes diseases. It induces a specific increase of the varicella antibodies. Thus varicella vaccination could prevent herpes recurrences in Sub-Saharan Africa. One- child dose varicella vaccine could be proposed as the first step to overcome HHV-3 herpes deficiency in order to prevent AIDS pandemic.

This work was supported in part through Battelle Memorial Institute?s prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I (J.H.K.). This work was also funded in part by National Institutes of Health (NIH) contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (R.B.T.) Rega Institute. Infectious Diseases unit. Leuven, Leuven, Belgium. United States Department of Agriculture. Agricultural Research Service. US Horticultural Research Laboratory. Fort Pierce, FL, USA. Ministry of Health of the Russian Federation. N. F. Gamaleya Federal Research Center for Epidemiology and Microbiology. D. I. Ivanovsky Institute of Virology. Moscow, Russia. University of Ljubljana. Ljubljana Faculty of Medicine. Ljubljana, Slovenia. Mississippi State University. Department of Biological Sciences. Mississippi State, MS, USA. University of Texas Medical Branch. Galveston, TX, USA. Institute of Diagnostic Virology. Friedrich-Loefer-Institut. Greifswald-Insel Riems, Germany. Centers for Disease Control and Prevention. Division of High-Consequence Pathogens and Pathology. Viral Special Pathogens Branch. Atlanta, GA, USA. Colorado State University. Department of Microbiology. Immunology & Pathology, Arthropod-borne and Infectious Diseases Laboratory. Fort Collins, CO, USA. Columbia University. Center for Infection and Immunity. Department of Epidemiology, Mailman School of Public Health. New York, NY, USA. University of California. Department of Molecular Biology and Biochemistry. Irvine, CA, USA. National Health Laboratory Service. Division of Virology. Bloemfontein. Republic of South Africa / University of the Free State. Division of Virology. Bloemfontein, Republic of South Africa. Colorado State University. Department of Microbiology. Immunology & Pathology, Arthropod-borne and Infectious Diseases Laboratory. Fort Collins, CO, USA. Unit? des Virus Emergents (Aix-Marseille Univ?IRD 190? Inserm 1207?IHU M?diterran?e Infection). Marseille, France. International Rice Research Institute. Plant Breeding Genetics and Biotechnology Division. Los Ba?os, Philippines. Les MandinauxLe Grand Madieu. France. The Scripps Research Institute. Department of Immunology and Microbiology IMM-6. La Jolla, USA. Unit? des Virus Emergents (Aix-Marseille Univ?IRD 190?Inserm 1207?IHU M?diterran?e Infection). Marseille, France. University of California. Department of Medicine. San Francisco, USA / University of California. Department of Biochemistry and Biophysics. San Francisco, USA / University of California. Department of Microbiology. San Francisco, USA. Istituto Agronomico Mediterraneo di Bari. Valenzano, Italy. Public Health Agency of Canada. National Microbiology Laboratory. Zoonotic Diseases and Special Pathogens. Winnipeg, Canada. Mayo Clinic. Department of Molecular Medicine. Rochester, USA. Istituto Agronomico Mediterraneo di Bari. Valenzano, Italy. Hacettepe University. Faculty of Medicine. Department of Medical Microbiology. Virology Unit. Ankara, Turkey. University of Texas Medical Branch. Galveston, TX, USA. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Chinese Center for Disease Control and Prevention. National Institute for Viral Disease Control and Prevention. Beijing, China. Kansas State University. Center of Excellence for Emerging and Zoonotic Animal Disease. Manhattan, USA. Chinese Center for Disease Control and Prevention. National Institute for Communicable Disease Control and Prevention. Beijing, China / Fudan University. Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences. Shanghai, China. WHO Collaborating Centre for Arboviruses and Hemorrhagic Fever Reference and Research. Bernhard-Nocht Institute for Tropical Medicine. Department of Virology. Hamburg, Germany. CNRS- Paris-Diderot. Institut Jacques Monod. Paris, France. The University of Queensland. School of Chemistry and Molecular Biosciences. Australian Infectious Diseases Research Centre. Brisbane, Australia. Public Health England. Salisbury, UK. Centers for Disease Control and Prevention. Fort Collins, USA. Indian Agricultural Research Institute. Division of Plant Pathology. New Delhi, India. Seoul National University. College of Agriculture and Life Sciences. Department of Agricultural Biotechnology, Center for Fungal Pathogenesis. Seoul, Korea. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / German Centre for Infection Research. Berlin, Germany. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / Slovak Academy of Sciences. Biomedical Research Center. Bratislava, Slovakia. Karolinska University Hospital. Center for Infectious Medicine, Karolinska Institutet. Department of Medicine Huddinge. Stockholm, Sweden. Wageningen University. Department of Plant Sciences. Laboratory of Virology. Wageningen, The Netherlands. Centers for Disease Control and Prevention. Fort Collins, USA. University of Washington. Department of Microbiology. Washington, USA. University of Louisville. School of Medicine. The Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases. Department of Pharmacology and Toxicology. Louisville, USA. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / German Centre for Infection Research. Berlin, Germany. University of Bari Aldo Moro. Department of Plant, Soil and Food Sciences. Bari, Italy. University of Hamburg. Biocentre Klein Flottbek. Hamburg, Germany. Folkhalsomyndigheten. Stockholm, Sweden. University of Hamburg. Biocentre Klein Flottbek. Hamburg, Germany. Washington State University. Irrigated Agricultural Research and Extension Center. Department of Plant Pathology. Prosser, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro de Inova??es Tecnol?gicas. Ananindeua, PA, Brasil. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Aristotle University of Thessaloniki. National Reference Centre for Arboviruses and Haemorrhagic Fever Viruses. Department of Microbiology, Medical School. Thessaloniki, Greece. National Health Laboratory Service. National Institute for Communicable Diseases. Centre for Emerging Zoonotic and Parasitic Diseases. Sandringham, South Africa / University of Pretoria. Centre for Viral Zoonoses, Faculty of Health Sciences. Department of Medical Virology. Pretoria South Africa. University of Texas Medical Branch. Galveston, TX, USA. University of Helsinki. Department of Virology. Medicum, Helsinki, Finland. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Universidade de Bras?lia. Departamento de Biologia Celular. Bras?lia , DF, Brazil. Universidad Nacional de La Plata - Consejo Nacional de Investigaciones Cient?ficas y T?cnicas. Centro Cientifico Technol?gico-La Plata. Instituto de Biotecnolog?a y Biolog?a Molecular. La Plata, Argentina. Institut Pasteur de Dakar. Dakar, Senegal. University of Maryland School of Medicine. Institute of Human Virology. Baltimore, USA. National Agriculture and Food Research Organization. Department of Planning and Coordination. Tsukuba, Japan. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. MRC-University of Glasgow Centre for Virus Research. Glasgow, UK. University of Tokyo. Asian Center for Bioresources and Environmental Sciences. Tokyo, Japan. University of Oxford. Department of Medicine. Oxford, UK. Bioinformatics Scientific Institute IRCCS E. MEDEA. Bosisio Parini, Italy. Korea University. College of Medicine. Department of Microbiology. Seoul. Republic of Korea. Centers for Disease Control and Prevention. Division of High-Consequence Pathogens and Pathology. Viral Special Pathogens Branch. Atlanta, GA, USA. Colorado State University. Immunology and Pathology. Department of Microbiology. Fort Collins, USA. University of Texas Medical Branch. Galveston, TX, USA. CNR. Institute for Sustainable Plant Protection. Torino, Italy. Fujian Agriculture and Forestry University. Institute of Plant Virology. Fujian Province Key Laboratory of Plant Virology. Fuzhou, China. North Carolina State University. Department of Entomology and Plant Pathology. Raleigh, USA. National Chung Hsing University. Department of Plant Pathology. Taichung, Taiwan. Universidade Federal de Vi?osa. Departamento de Fitopatologia/BIOAGRO. Vi?osa, MG, Brazil. Chinese Center for Disease Control and Prevention. National Institute for Communicable Disease Control and Prevention. Beijing, China / Fudan University. Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences. Shanghai, China. Chinese Academy of Agricultural Sciences. Institute of Plant Protection. State Key Laboratory for Biology of Plant Diseases and Insect Pests. Beijing, China. National Institutes of Health (NIH). National Institute of Allergy and Infectious Diseases (NIAID). Division of Clinical Research (DCR). Integrated Research Facility at Fort Detrick (IRF-Frederick). Frederick, USA. In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Introduction: At the onset of the 20th century, ancient clinical observations of cancer epidemics in Bantu populations of Sub-Saharan Africa were discovered. They were reported from 1914 to 1960, but remained unexplained. In 1983, in San Francisco, Calif., USA, cancer epidemics were related to infections by the human immunodeficiency virus type 1 (HIV-1) known as AIDS disease. Yet since 1996, it is known that HIV-1 strains are not the only ones involved. In Sub-Saharan Africa, recurrent orobuccal herpes simplex virus type 1 (HSV-1) and genital recurrent herpes simplex virus type 2 (HSV-2) appeared many times prior to infection by HIV-1. Case Reports: Data on these ancient medical observations regarding African cancer epidemics can today be referred to as the relationship between the unfortunate immune deficiency of herpes in Bantu populations and HIV-1 viral strains. For centuries, the Bantu populations dispersed in forests were living in close proximity to chimpanzees infected by simian immunodeficiency virus (SIV) and were exposed to SIV contamination which became HIV-1 in human beings. Presently, these unexplained Bantu cancer epidemics can be linked to the viral partnership of HSV-1/HSV-2 to HIV-1 strains. Conclusion: The key issue is now to prevent HSV-1/HSV-2 diseases related to HIV-1. An anti-herpes treatment administered early during childhood to Bantu populations will offer a mean of preventing herpes diseases related to HIV-1 infection and hence avoid cancer epidemics.

The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5′-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5′ cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.

The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA. To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins.

The first ∼60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region. In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro. Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment.

Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5′ region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro.

The role of the 3' noncoding (NC) region of Physalis mottle tymovirus genomic RNA in the multiplication of the virus was examined using an in vivo protoplast assay system. Coat protein (CP) synthesis was specifically inhibited by sense 3' NC region transcripts. To establish the role of the pseudoknot structure present in the NC region in virus multiplication, four site-specific mutants, two of which disrupted the pseudoknot structure while the other two restored the structure, were constructed. Interestingly, none of the four sense mutant transcripts inhibited CP synthesis, suggesting that the specific sequence representing the 3' terminal pseudoknot structure may play an important role in virus multiplication. However, the wild-type antisense 3' NC transcript as well as the four antisense mutant transcripts inhibited CP synthesis, suggesting that the inhibitions by antisense transcripts could be due to the formation of RNA-RNA hybrids at the 3' end of the genomic RNA.

The first demonstration on the aminoacylation capacity of the RNA genome of a plant virus appeared more than 25 years ago. Shortly thereafter, aminoacylation of the RNA genome of a number of other plant viruses was observed. This led to considerable work on the tRNA-like region of these viral RNAs, and to the first demonstration of the presence of pseudoknots in their folding pattern. In spite of the vast amount of efforts put into trying to understand the reason for the aminoacylation capacity of certain viral RNA genomes, as yet no clear general conclusion emerges. It rather looks as though the reason for aminoacylation may be different for different viruses, and that aminoacylation may operate at different levels in the virus life cycle. Given that certain RNA viruses possess structures which resemble that of tRNAs at their 5'- or 3'-termini, it is most likely that convergent evolution may have dominated the appearance of such structures in the virus world.

Introduction. Most plant viruses contain an RNA genome and are traditionally classified into genera (or families) of which there are over 40. This large number reflects the formidable variety that exists among plant RNA viruses. The genomes are mostly single-stranded (ss) and of positive polarity; in a few genera, they are negative sense, ambisense, or double-stranded (ds). The different strategies used by viruses for their amplification have been described at length. However, it is often difficult to find in a summarized form the main data on genome features of all groups of plant RNA viruses. The last review article along these lines (Davies & Hull, 1982) dates from before any full-length RNA sequence of a plant RNA virus was published; never-theless, a few books have more recently included this subject (Francki et al., 1991; Matthews, 1991).

Sequence comparisons predicted a potential papain-like proteinase domain in the N-terminal cleavage product (NRP) of the large nonstructural replicase polyprotein (RP) of turnip yellow mosaic virus (TYMV). Replacement of the predicted catalytic amino acids, Cys-783 by Ser, or of His-869 by Glu, abolished cleavage of the 206K RP into a approximately 150 K NRP and a approximately 78 K C-terminal product in reticulocyte lysates, while other substitutions exerted no apparent influence on proteolysis. The proteinase-deficient mutant RPs could not be cleaved in trans by as much as an eight-fold molar excess of wild-type proteinase. Deletion experiments have excluded the possible influence on autoproteolysis of amino acid sequences 1-708 and 982-1204 flanking the proteinase domain. Thus, the proteinase of TYMV with a papain-like dyad of essential amino acids has been mapped just upstream from the putative NTPase domain. Statistically significant sequence similarities with the TYMV proteinase were found for the similarly located domains of the replicase polyproteins of carlaviruses, capilloviruses, apple stem pitting virus and apple chlorotic leaf spot virus as well as for those of other tymoviruses and for the domain located downstream from the putative NTPase domain of the large polyprotein of beet necrotic yellow vein furovirus. All these domains are not significantly similar to other known proteinases, although they conserve papain-like Cys- and His-containing motifs. Thus these domains constitute a compact group of related enzymes, the tymo-like proteinases, within the proposed papain-like proteinase supergroup. The resulting alignment of 10 tymo-like proteinase sequences has revealed a third highly conserved residue--Gly (Gly821 in TYMV RP) followed by a hydrophobic residue. We speculate that all the tymo-like proteinase domains of the viral replicative proteins may share common biochemical and biological features.

Recently, cDNA clones encoding the bovine (b) [M. Garret, B. Pajot, V. Trezeguet, J. Labouesse, M. Merle, J.-C. Gandar, J.-C. Gandar, J.-P. Benedetto, M.-L. Sallafranque, J. Alterio, M. Gueguen, C. Sarger, B. Labouesses and J. Bonnet (1991) Biochemistry 30, 7809–7817] and human (h) [L. Yu. Frolova, M. A. Sudomoina, A. Yu. Grigorieva, O. L. Zinovieva and L. L. Kisselev (1991) Gene 109, 291–296] tryptophanyl-tRNA synthetases (TrpRS) were sequnced; the deduced amino acid sequences exhibit typical structural features of class I aminoacyl-tRNA synthetases [G. Eriani, M. Delarue, O. Poch, J. Gangloff and D. Moras (1990) Nature 237, 203–206] and limited, although significant, similarity with bacterial TrpRS. Independently, it was shown that a major protein whose synthesis is stimulated in human cell cultures by interferon γ [J. Fleckner, H. H. Rasmussen and J. Justesen (1991) Proc. Natl Acad. Sci. USA 88, 11 520–11 524], and interferons γ or α [B. Y. Rubin, S. L. Anderson, L. Xing, R. J. Powell and W. P. Tate (1991) J. Biol. Chem. 226, 24 245–24 248], exhibits TrpRS activity and an amino acid sequence identical to that of hTrpRS. The amino acid sequences of bTrpRS and hTrpRS are highly similar and are surprisingly very similar to the amino acid sequence deduced from a cloned and sequenced cDNA reported to encode rabbit (r) peptide-chain-release factor (RF) [C. C. Lee, W. J. Craigen, D. M. Muzny, E. Harlow and C. T. Caskey (1990) Proc. Natl Acad. Sci. USA 87, 3508–3512]. This close similarity between mammalian TrpRS and cloned RF is unexpected given the distinct functional properties of these proteins. Consequently, the question arises as to whether the mammalian TrpRS and RF activities reside on identical or very similar polypeptides. Alternatively, one may assume that the cloned rabbit cDNA encodes a protein other than rRF. Several properties (immunochemical, biochemical and physico-chemical) of mammalian TrpRS and RF have been compared. rTrpRS and rRF have distinct thermostabiliy behaviours, and dissimilar chromatographic profiles on phosphocellulose. Both the anti-bTrpRS polyclonal antibodies and the monoclonal antibody Am2 strongly inhibit the bTrpRS and hTrpRS aminoacylation activities, but not the rRF activity. In addition, neither bTrpRS nor hTrpRS exhibit RF activity. It is concluded that (a) the functional centers responsible for the TrpRS and RF activities are structurally and functionally dissimilar and could hardly belong to the same protein domain; (b) RF and TrpRS probably reside on different polypeptide chains encoded by different genes, although it is still not excluded that the RF- and TrpRS-specific domains are situated on one and the same polypeptide chain; (c) the rabbit cDNA believed to encode RF, presumably encodes rTrpRS; this would explain why its deduced amino acid sequence has no similarity with bacterial RFs but is very similar to mammalian TrpRS.

No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.

International audience; Regulation of protein synthesis by viruses occurs at all levels of translation. Even prior to protein synthesis itself, the accessibility of the various open reading frames contained in the viral genome is precisely controlled. Eukaryotic viruses resort to a vast array of strategies to divert the translation machinery in their favor, in particular, at initiation of translation. These strategies are not only designed to circumvent strategies common to cell protein synthesis in eukaryotes, but as revealed more recently, they also aim at modifying or damaging cell factors, the virus having the capacity to multiply in the absence of these factors. In addition to unraveling mechanisms that may constitute new targets in view of controlling virus diseases, viruses constitute incomparably useful tools to gain in-depth knowledge on a multitude of cell pathways.

Publisher Summary Virus‐like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. The chapter outlines the recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are: (1) The requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. VLP technology is a rapidly advancing domain of molecular and structural biology. Extensive progress in VLP studies was achieved as the insect cell based protein production system was developed. This baculovirus expression system has many advantages for the synthesis of viral structural proteins resulting in the formation of VLPs. It allows production of large amounts of correctly folded proteins while also providing cell membranes that can serve as structural elements for enveloped viruses. These features give us the opportunity to gain insights into the interactions and requirements accompanying VLP formation that are similar to the assembly events occurring in mammalian cells. Other encouraging elements are the ability to easily scale up the system and the simplicity of purification of the assembled VLPs. The growing number of VLPs carrying foreign protein fragments on their surface and studies on the successful assembly of these chimeric molecules is a promising avenue towards the development of a new technology, in which the newly designed VLPs will be directed to particular mammalian cell types by exposing specific binding domains. The progress made in modeling the surface of VLPs makes them to date the best candidates for the design of delivery systems that can efficiently reach their targets.

Virus‐like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. The chapter outlines the recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are: (1) The requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. VLP technology is a rapidly advancing domain of molecular and structural biology. Extensive progress in VLP studies was achieved as the insect cell based protein production system was developed. This baculovirus expression system has many advantages for the synthesis of viral structural proteins resulting in the formation of VLPs. It allows production of large amounts of correctly folded proteins while also providing cell membranes that can serve as structural elements for enveloped viruses. These features give us the opportunity to gain insights into the interactions and requirements accompanying VLP formation that are similar to the assembly events occurring in mammalian cells. Other encouraging elements are the ability to easily scale up the system and the simplicity of purification of the assembled VLPs. The growing number of VLPs carrying foreign protein fragments on their surface and studies on the successful assembly of these chimeric molecules is a promising avenue towards the development of a new technology, in which the newly designed VLPs will be directed to particular mammalian cell types by exposing specific binding domains. The progress made in modeling the surface of VLPs makes them to date the best candidates for the design of delivery systems that can efficiently reach their targets.

Publisher Summary This chapter provides an updated view of the host factors that are, at present, believed to participate in replication/transcription of RNA viruses. One of the major hurdles faced when attempting to identify host factors specifically involved in viral RNA replication/transcription is how to discriminate these factors from those involved in translation. Several of the host factors shown to affect viral RNA synthesis are factors known to be involved in protein synthesis, for example, translation factors. In addition, some of the factors identified to date appear to influence viral RNA amplification as well as viral protein synthesis, and translation and replication are frequently tightly associated. Several specific host factors actively participating in viral RNA transcription/replication have been identified and the regions of host protein/replicase or host protein/viral RNA interaction have been determined. The chapter centers exclusively on those factors that appear functionally important for viral amplification. It presents a list of the viruses for which a specific host factor associates with the polymerase, affecting viral genome amplification. It also indicates the usually accepted cell function of the factor and the viral polymerase or polymerase subunit to which the host factor binds.

A biochemical world that would have existed before the contemporary DNA-RNA-Protein world, and baptized in 1986 "The RNA World" by Walter Gilbert, such a world had already been proposed during the preceding decades by Carl Woese, Francis Crick and Leslie Orgel. By demonstrating the remarkable diversity of the RNA molecule, Molecular Biology proved these predictions. RNA present in all living cells, performs structural and metabolic functions many of which were unsuspected only a few years ago. A truly modern "RNA world" exists in each cell; it contains RNAs in various forms, short and long fragments, single and double-stranded, endowed with multiple roles (informational, catalytic, that can serve as templates, guides, defense), certain molecules being even capable of carrying out several of these functions.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1•eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1•eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Introduction. The genus Potyvirus, family Potyviridae, is the largest genus of plant viruses with 180 members or possible members (Brunt, 1992). Potyviruses are flexuous filamentous particles which contain a single-stranded RNA genome of positive polarity possessing a covalently linked 5′-terminal viral protein (VPg) and a 3′-terminal poly(A) tail (reviewed in Riechmann et al., 1992). They are transmitted from plant to plant by aphids in a non-persistent manner, and this process is dependent on the presence of two virus-encoded proteins (reviewed in Pirone, 1991). One of these, the helper component-proteinase (HC-Pro) has attracted renewed attention during the last few years due to its multifunctionality and to it being implicated in different steps of the potyvirus life cycle. The properties, as well as the established and postulated functions of this protein, are reviewed.

The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C- terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.

THE termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established1–4, that of the only known eukaryotic RF (eRF)5 remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRFl) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human6 and the other from Xenopus laevis7 , have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (supl) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation8–12. The amino-acid sequence of the eRFl family is highly conserved. We conclude that the eRFl proteins are directly implicated in the termination of translation in eukaryotes.

Introduction. The tenuiviruses are a most unusual type of virus about which little was known until recently. They were officially recognized as a plant virus group in 1983 (reviewed in Gingery, 1988). They are described in the Fifth Report of the International Committee on Taxonomy of Viruses as non-enveloped plant viruses, with possibly a negative ssRNA genome (Francki et al., 1991). Five viruses belong to this group, the type member rice stripe virus (RSV) first discovered in Japan in the 1890s, followed by maize stripe virus (MStV) in Mauritius in 1929, rice hoja blanca virus (RHBV) in Colombia in 1935, European wheat striate mosaic virus (EWSMV) in England in 1956, and rice grassy stunt virus (RGSV) in the Philippines in 1963 (reviewed in Gingery, 1988). Epidemics of RSV and RHBV cause important yield losses in rice-growing areas of Asia and the former U.S.S.R. (Toriyama, 1983), and of tropical America (Morales & Niessen, 1985) respectively.

In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3′-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region. The two chimeric genes were then introduced into the genome of rapeseed ( Brassica napus ) using an Agrobacterium rhizogenes vector system. Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions. One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected. Protection was overcome when the inoculum concentration was increased. RNA complementary to the initial transcript was detected after infection.

The complete sequence of rice hoja blanca virus (RHBV) RNA4 has been determined, based on the sequence of the corresponding cDNA clones. RNA4 consists of 1991 nucleotides with two open reading frames (ORFs). One putative ORF is located in the 5'-proximal region of the viral RNA4; it encodes a protein of predicted M(r) 20076 which corresponds to the major non-structural protein that accumulates in RHBV-infected rice plants, and which bears limited sequence identity with the helper component of tobacco vein mottling potyvirus. The other ORF is located in the 5'-proximal region of the viral complementary RNA4 and encodes a protein of predicted M(r) 32,469. Between the two ORFs is an intergenic region of 524 nucleotides, part of which can theoretically adopt a stable stem-loop structure; the 5' and 3' ends can potentially base-pair over 16 nucleotides, producing a pan-handle configuration. These characteristics are in favour of an ambisense coding strategy for RHBV RNA4.

A very high (approximately 90%) structural similarity exists between the bovine, human and murine tryptophanyl-tRNA synthetases (WRS), and quite unexpectedly the rabbit polypeptide chain release factor (eRF). This similarity may point to a very close resemblance or identity between these proteins involved in distinct steps of protein synthesis, or inadvertently to an incorrect assignment of the clone reported to encode eRF, since the structure of clones encoding WRS were confirmed by peptide sequencing. Using high resolution column chromatography and sucrose gradient centrifugation combined with assays for WRS and eRF activities, we show that functionally distinct WRS and eRF proteins can be completely separated from each other. Moreover, a putative anti-eRF monoclonal antibody appears incapable of immunoprecipitating the eRF activity or binding to protein(s) possessing eRF activity. This antibody binds to protein fractions which coincide in various separation procedures with rabbit WRS activity, and to pure bovine WRS. The protein expressed in Escherichia coli from the original cDNA clone initially reported to encode eRF, has WRS activity but not eRF activity. Resequencing of the fragment of the original rabbit cDNA demonstrates the presence of the previously overlooked HXGH motif typical of class I aminoacyl-tRNA synthetases. Consequently, mammalian WRS and eRF are different proteins, and the cDNA clone formerly assigned as encoding eRF encodes rabbit WRS.

We have previously shown that the Dictyostelium discoideum ribosomal protein L19 specifically binds Ca2+/calmodulin [Sonneman et al. (1991) J. Biol. Chem. 266, 23091-23096]. To investigate the role of calmodulin in the regulation of protein synthesis, we have now established an in vitro protein synthesizing system from Dictyostelium cells which can elongate polypeptide chains with high efficiency. Various calmodulin antagonists affected translation in this system. The inhibitory effects of the antagonists could be partially reversed by addition of calmodulin. A monoclonal antibody against D. discoideum calmodulin also specifically inhibited protein synthesis. Similar effects of calmodulin antagonists were found in a standard wheat germ in vitro translation system.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).

We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1.5 x 10(6) rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.

Total nucleotide sequencing of the RNA genome of various tymoviruses has demonstrated that the overall genome organization of these viruses is identical. Furthermore, the strategies of expression of the turnip yellow mosaic virus (TYMV) genome have been established by in vitro translation studies; these include the synthesis of a subgenomic RNA, the utilization of overlapping open reading frames (ORFs) and maturation of a polyprotein. In the experiments described here, the strategies of expression of other tymovirus (eggplant mosaic virus, ononis yellow mosaic virus, belladonna mottle virus and physalis mottle virus) genomes have been compared to those used by the TYMV genome, in particular to determine whether these tymoviruses also resort to the expression of overlapping ORFs and maturation of a polyprotein.

We have previously shown that the last 100 nucleotides from the 3' end of turnip yellow mosaic virus (TYMV) RNA compete in vitro with genomic RNA for the TYMV-specific RNA-dependent RNA polymerase (RdRp). To further characterize the promoter on genomic RNA that produces complementary RNA strands, shorter fragments corresponding to the 3' region of the viral RNA were generated and used in in vitro assays. Fragments as short as 38 nucleotides corresponding to the 3' end of TYMV RNA compete with the viral RNA for the RdRp suggesting that the 3' promoter on plus strand RNA is probably less than or equal to 38 nucleotides long. These transcripts are themselves used as templates in vitro.

En moins de trois ans, une dizaine d'etudes fondees sur la diffraction de cristaux aux rayons X, sur la resonance magnetique nucleaire et sur la cryomicroscopie electronique ont permis d'obtenir une resolution a l'echelle atomique de la structure du ribosome bacterien et de comprendre les bases moleculaires de la synthese des proteines. Il s'en degage que l'ARN ribosomique joue un role preponderant dans la structure du ribosome ainsi que dans la formation de la liaison peptidique et le decodage du code genetique. Ces resultats ouvrent la voie a la conception de nouveaux antibiotiques diriges contre le ribosome bacterien et a d'autres therapeutiques ayant pour cible la synthese proteique.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.

The RNA conformational requirements for both aminoacylation and adenylation emerging from our studies performed using the valine- and the tyrosine-accepting plant viral RNAs are now strongly supported by the histidine-accepting tobacco mosaic virus RNA: an 'L'-shaped conformation is recognized by the aminoacyl-tRNA synthetase whereas only the aminoacyl RNA domain (equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop) interacts with the tRNA nucleotidyltransferase.

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure as an integral part of its genome. This structure is located at the extreme 3' end of the viral RNA and is the acceptor of valine after 3'-terminal adenylation. It is known that in vitro (with bacterial, yeast, or plant systems) and in vivo (upon microinjection into Xenopus laevis oocytes) a series of tRNA-specific enzymes can recognize this structure in the viral RNA. We report that TYMV RNA is valylated and consequently adenylated in vivo in its natural host, Chinese cabbage leaves. This suggests that the acylated form of the viral RNA could play an important role in the life-cycle of the virus.

RNA extracted from ovaries of adult Xenopus laevis and introduced into a wheat germ cell-free system, directs the synthesis of polypeptides co-electrophoresing with all five histones. By frationation of total ovary RNA through Sephadex G-200 and polyacrylamide gel, RNA was isolated that is translated into histones 4 (H4) only. In addition, this RNA labelled in vitro with [I125] hybridizes to a restriction fragment of cloned sea urchin DNA known to contain the H4 gene.

The genomic RNA of turnip yellow mosaic virus is translated in vitro into two major high-molecular-weight proteins, the larger of which (Mr 195 000) undergoes post-translational cleavage. The mechanism of formation of the primary cleavage products (Mr 120 000 and Mr 78 000) of the 195 000-Mr protein has been examined. The fact that cleavage partly occurs at a rate insensitive to dilution of the 195 000-Mr protein is suggestive of an intramolecular mechanism of proteolytic maturation.

Tobacco mosaic virus and turnip yellow mosaic virus genome RNAs, which have long (greater than or equal to 69 nucleotides) 5'-terminal leader sequences preceding the AUG initiation codon, can form disome initiation complexes when incubated with a wheat germ extract and sparsomycin. In tobacco mosaic virus RNA, whereas one ribosome in such complexes presumably occupies the AUG-containing initiation site, the other is located within the RNA leader sequence. The results favor a model of eukaryotic initiation in which ribosomes first bind to the 5' end of the mRNA independently of AUG, then advance to the AUG initiation codon, where protein synthesis ensues.

The genome of most plant viruses is composed of RNA of ‘ + ’ polarity and can serve directly as messenger for protein synthesis. This paper deals with those viral RNAs whose in vitro and in vivo translation products can be compared; it reviews the strategies used for the modulation of the synthesis of the various virus-coded proteins and examines the postulated functions of these proteins. Plant RNA virus In vitro translation In vivo translation Protein synthesis regulation

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.

Two amber suppressor tRNAs have been isolated from calf liver. They are different from previously identified naturally occurring amber suppressors of eukaryotes in so far as they are neither tRNATyr nor tRNAGln. They are leucine iso-acceptors and their nucleotide sequence indicates that they harbour a CAA and a CAG anticodon respectively. Both species are functional as amber suppressors as demonstrated by readthrough of the amber codon which terminates the 126 kd protein gene of tobacco mosaic virus RNA. The results bring new information in the discussion of codon-anticodon recognition and regulation of termination in eukaryotic protein synthesis.

This paper describes the minimum length of the turnip yellow mosaic virus (TYMV) RNA necessary to fulfill the tRNA-like properties of the viral RNA: 50 to 75 nucleotides and 86 nucleotides from the 3' end of TYMV RNA are sufficient for adenylation and valylation respectively by the Escherichia coli system. The size of the tRNA-like fragments obtained in vitro in the presence of an E. coli, a reticulocyte or a chinese cabbage leaf extract has also been determined. Among the major fragments liberated from the 3' end of TYMV RNA by the three systems are fragments of 117 and 112 nucleotides. In addition, the E. coli extract liberates fragments of 139 and 61 nucleotides, and the reticulocyte lysate fragments of 109, 94, 84, 73 and 46 nucleotides. The cleavage of the viral RNA by several systems in vitro to yield RNA fragments encompassing the tRNA-like sequence suggests that such fragments might also be liberated in vivo.

Summary In particles of turnip yellow mosaic virus (TYMV), the interactions between proteins are particularly strong when compared to those between proteins in some other icosahedral viruses. Intact RNA is released from TYMV particles on freezing and this process has been studied by examining several parameters that influence cryodenaturation such as dehydration, pressure, aggregation and the presence of protective agents (glycerol and ammonium sulphate). Pressure of 1.5 × 108 Pa had no effect on virus particles whereas dehydration of a virus suspension had a drastic effect. Aggregate formation resulting from freezing of solutions containing high virus concentrations seems to be a prerequisite for RNA release; cryoprotective agents hindered RNA release.

The 3'-terminal sequence of tomato aspermy virus (TAV, a cucumovirus) RNA can be arranged in an 'L'shaped conformation similar to that of the 3'-tRNA-like regions of the tyrosine-accepting bromo-, cucumoand hordeivirus RNAs examined to date. We report here that TAV RNA is recognized by the tRNA nucleotidyltransferase and can be adenylated nearly as efficiently as brome mosaic virus (BMV, a bromovirus) RNA. However, using tyrosyl-tRNA synthetase from rat liver or from yeast, TAV RNA cannot be tyrosylated under conditions that allow aminoacylation of BMV RNA. Structural features in the 3'-region could be responsible for the recognitory discrimination between the BMV and TAV RNAs by these eukaryotic tyrosyl-tRNA synthetases.