Structure of deformed wing virus, a major honey bee pathogen.

The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism.

Viruses from the family Iflaviridae are insect pathogens. Many of them, including slow bee paralysis virus (SBPV), cause lethal diseases in honeybees and bumblebees, resulting in agricultural losses. Iflaviruses have nonenveloped icosahedral virions containing single-stranded RNA genomes. However, their genome release mechanism is unknown. Here, we show that low pH promotes SBPV genome release, indicating that the virus may use endosomes to enter host cells. We used cryo-EM to study a heterogeneous population of SBPV virions at pH 5.5. We determined the structures of SBPV particles before and after genome release to resolutions of 3.3 and 3.4 Å, respectively. The capsids of SBPV virions in low pH are not expanded. Thus, SBPV does not appear to form "altered" particles with pores in their capsids before genome release, as is the case in many related picornaviruses. The egress of the genome from SBPV virions is associated with a loss of interpentamer contacts mediated by N-terminal arms of VP2 capsid proteins, which result in the expansion of the capsid. Pores that are 7 Å in diameter form around icosahedral threefold symmetry axes. We speculate that they serve as channels for the genome release. Our findings provide an atomic-level characterization of the genome release mechanism of iflaviruses.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

Polyhedra structures and the evolution of the insect viruses.

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.

CapsidMaps: protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps.

Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.

Dynamic and geometric analyses of Nudaurelia capensis ω virus maturation reveal the energy landscape of particle transitions.

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity.

Characterization of Port Bolivar Virus, a Novel Entomobirnavirus (Birnaviridae) Isolated from Mosquitoes Collected in East Texas, USA.

This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of Aedes sollicitans mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus Entomobirnavirus, family Birnaviridae. PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Comparison of microscopic and molecular enumeration methods for insect viruses: Cryptophlebia leucotreta granulovirus as a case study.

Enumeration techniques were compared for quantification of the South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA), used as a biopesticide to control false codling moth (Thaumatotibia leucotreta), an insect pest of various fruits and nuts, including citrus. The routine enumeration method for CrleGV-SA virus particles in experimentation and production of CrleGV-SA biopesticides is dark field microscopy. This method was compared with spectrophotometry, scanning electron microscopy (SEM) and real time quantitative polymerase chain reaction (qPCR). The purpose was to develop an accurate and reliable routine enumeration method for CrleGV-SA occlusion bodies (OBs) and to validate the use of dark field microscopy. Purified and semi-purified CrleGV-SA viral stocks were used. Spectrophotometry was not a suitable or accurate enumeration method. Dark field microscopy and SEM were accurate and statistically comparable (p = 0.064), validating the use of dark field microscopy as an enumeration method for granulovirus (GV). However, SEM has superior resolution and the advantage of easily distinguishing virus particles from debris in semi-purified viral stock preparations. A quantitative PCR technique has been developed based on use of specific oligonucleotide primers for the granulin gene. This has the advantage of not being affected by contamination with non-biological debris or biological material, which impact on the other methods.

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography.

Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections.

Artifical transfer and morphological description of virus particles associated with superparasitism behaviour in a parasitoid wasp.

In parasitoids, the adaptive significance of superparasitism (laying of egg(s) in already parasitized hosts) has been the subject of strong controversy. The current view is to interpret this behaviour as an adaptation to increased competition for hosts, because the supernumerary egg still has a chance to win possession for the host. However, we recently discovered that in the solitary parasitoid Leptopilina boulardi, superparasitism is rather caused by an unknown infectious element: stable non superparasitizing lineages (NS) are transformed into stable superparasitizing lineages (S) after eggs from both lineages have competed inside the same host (superparasitism). In this report, we investigate the nature and location of the causative agent. Involvement of bacteria is unlikely because antibiotic treatments do not affect wasp phenotype and because bacterial 16S ribosomal DNA was not detected using PCR. We report successful injection experiments showing that the causative agents are located in wasp poison gland and ovaries and are stably inherited. Electron microscopic studies demonstrate that long filamentous virus particles located in wasp oviducts are strongly associated with superparasitism behaviour, leading to reconsider the adaptive significance of this behaviour in parasitoids. Interestingly, parasitoids are often infected with similar viruses for which no phenotypic effect has been documented. This raises the possibility that they could induce the same behavioural manipulation.

Functional implications of quasi-equivalence in a T = 3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography.

BACKGROUND: Studies of simple RNA animal viruses show that cell attachment, particle destabilization and cell entry are complex processes requiring a level of capsid sophistication that is difficult to achieve with a shell containing only a single gene product. Nodaviruses [such as Flock House virus (FHV)] are an exception. We have previously determined the structure of FHV at 3 A resolution, and now combine this information with data from cryo-electron microscopy in an attempt to clarify the process by which nodaviruses infect animal cells. RESULTS: A difference map was computed in which electron density at 22 A resolution, derived from the 3.0 A resolution X-ray model of the FHV capsid protein, was subtracted from the electron density derived from the cryo-electron microscopy reconstruction of FHV at 22 A resolution. Comparisons of this density with the X-ray model showed that quasi-equivalent regions of identical polypeptide sequences have markedly different interactions with the bulk RNA density. Previously reported biphasic kinetics of particle maturation and the requirement of subunit cleavage for particle infectivity are consistent with these results. CONCLUSIONS: On the basis of this study we propose a model for nodavirus infection that is conceptually similar to that proposed for poliovirus but differs from it in detail. The constraints of a single protein type in the capsid lead to a noteworthy use of quasi-symmetry not only to control the binding of a 'pocket factor' but also to modulate maturation cleavage and to release a pentameric helical bundle (with genomic RNA attached) that may further interact with the cell membrane.

Binding and entry of a non-enveloped T=4 insect RNA virus is triggered by alkaline pH.

Tetraviruses are small, non-enveloped, RNA viruses that exclusively infect lepidopteran insects. Their particles comprise 240 copies of a single capsid protein precursor (CP), which undergoes autoproteolytic cleavage during maturation. The molecular mechanisms of capsid assembly and maturation are well understood, but little is known about the viral infectious lifecycle due to a lack of tissue culture cell lines that are susceptible to tetravirus infection. We show here that binding and entry of the alphatetravirus, Helicoverpa armigera stunt virus (HaSV), is triggered by alkaline pH. At pH 9.0, wild-type HaSV virus particles undergo conformational changes that induce membrane-lytic activity and binding to Spodoptera frugiperda Sf9 cells. Binding is followed by entry and infection, with virus replication complexes detected by immunofluorescence microscopy within 2h post-infection and the CP after 12h. HaSV particles produced in S. frugiperda Sf9 cells are infectious. Helicoverpa armigera larval virus biofeed assays showed that pre-treatment with the V-ATPase inhibitor, Bafilomycin A1, resulted in a 50% decrease in larval mortality and stunting, while incubation of virus particles at pH 9.0 prior to infection restored infectivity. Together, these data show that HaSV, and likely other tetraviruses, requires the alkaline environment of the lepidopteran larval midgut for binding and entry into host cells.

Analysis of rapid, large-scale protein quaternary structural changes: time-resolved X-ray solution scattering of Nudaurelia capensis omega virus (NomegaV) maturation.

Time-resolved small-angle X-ray scattering (TR-SAXS) was used to study the kinetics of a large conformational change that occurs during the maturation of an icosahedral virus. Virus-like particles (VLPs) of the T=4 non-enveloped RNA virus Nudaurelia capensis omega virus (NomegaV) were shown to undergo a large pH-dependent conformational change. Electron cryo-microscopy (cryoEM) and X-ray solution scattering were used to show that the precursor VLP (procapsid) was 16 % larger in diameter than the resulting capsid, which was shown by the cryoEM study to closely resemble the infectious mature virion. The procapsid form of the VLPs was observed at pH 7.5 and was converted to the capsid form at pH 5.0. Static SAXS measurements of the VLPs in solutions ranging between these pH values determined that the half-titration point of the transition was pH 6.0. Time-resolved SAXS experiments were performed on VLP solutions by initiating a pH change from 7.5 to 5.0 using a stopped-flow device, and the time-scale of the conformational change occurred in the subsecond range. Using a less drastic pH change (lowering the pH to 5.8 or 5.5), the conformational change occurred more slowly, on the subminute or minute time-scale, with the detection of a fast-forming intermediate in the transition. Further characterization using static SAXS measurements showed that the conformational change was initially reversible but became irreversible after autoproteolytic maturation was about 15 % complete. In addition to characterizing the large quaternary conformational change, we have been able for the first time to demonstrate that it takes place on the subsecond time-scale, a regime comparable to that observed in other multisubunit assemblies.

The 2.8 A structure of a T = 4 animal virus and its implications for membrane translocation of RNA.

Simple RNA animal viruses generally enter cells through receptor-mediated endocytosis followed by acid pH dependent release and translocation of RNA across the endosomal membrane. The T = 3 nodaviruses contain prefabricated pentameric helical bundles that are cleaved from the remainder of the subunits by an assembly-dependent auto-proteolysis and they are positioned for release through 5-fold axes of the particle. We previously proposed that these bundles may serve as conduits for RNA membrane translocation. Additional support for this hypothesis is now provided by the first atomic resolution structure of a T = 4 RNA virus, where we find cleavage sites and helical bundles nearly identical with those observed in T = 3 nodaviruses. The helices are of sufficient length to span a membrane bilayer and the internal diameter of the coiled bundle could accommodate ssRNA. The T = 4 particle has a mean outer diameter of 410 A and is formed by 240 copies of a single subunit type. The subunit is composed of a helical inner domain (where the cleavage occurs) containing residues preceding and following a canonical, viral, eight-stranded beta-sandwich that forms the contiguous shell. Inserted between two strands of the shell domain are 133 residues with an immunoglobulin c-type fold. The initial gene product consists of 644 amino acid residues and is cleaved between residues Asn570 and Phe571 in the mature particle determined in this analysis.

Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV).

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.

RNA 1 and RNA 2 Genomic Segments of Chronic Bee Paralysis Virus Are Infectious and Induce Chronic Bee Paralysis Disease.

Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1 (3,674 nt) and RNA 2 (2,305 nt). Three minor RNAs (about 1,000 nt each) have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of in vivo inoculation of the two purified CBPV major RNAs are presented and demonstrate that RNA 1 and RNA 2 are infectious. Honeybees inoculated with 10(9) RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV.

Unusual morphology of a virus which produces carbon dioxide sensitivity in mosquitoes.

A virus, named Matsu, presumed to be the etiologic agent of hereditary sensitivity to carbon dioxide in Culex quinquefasciatus mosquitoes, was adapted to growth in the C6/36 line of Aedes albopictus cells. Though it was expected that the mosquito virus would be a rhabdovirus like sigma, the etiologic agent of hereditary carbon dioxide sensitivity in Drosophila melanogaster flies, that was not the case. The virion of Matsu was found to be unlike any previously described virus. It was pleomorphic, enveloped, from 200 to 550 nm in maximum diameter, and contained from three to several dozen virus-like polyhedral structures approximately 30 nm in diameter.

Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus.

Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.