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PubMed1999

The capsid gene of feline calicivirus contains linear B-cell epitopes in both variable and conserved regions.

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.

Amino Acid Sequence↗
PubMed2000

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport.

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.

Animals↗
PubMed2000

Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape.

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

Amino Acids↗
PubMed2000

Proteolytic processing of the astrovirus capsid.

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35S]Smethionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10(5)-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.

Antigens, Viral↗
PubMed2001

A monoclonal antibody specific for reovirus outer-capsid protein sigma3 inhibits sigma1-mediated hemagglutination by steric hindrance.

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.

Animals↗
PubMed2001

Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

Capsid↗
PubMed2001

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins.

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.

Blotting, Western↗
PubMed2003

Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation.

The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.

Capsid↗
PubMed2003

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

Amino Acid Sequence↗
PubMed2003

A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein.

Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.

Amino Acid Sequence↗
PubMed2004

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Adenoviridae↗
PubMed1984

Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.

Capsid↗
PubMed2005

Engineering thermal stability in RNA phage capsids via disulphide bonds.

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.

Allolevivirus↗
PubMed2004

Competing hydrophobic and screened-coulomb interactions in hepatitis B virus capsid assembly.

Recent experiments show that, in the range from approximately 15 to 45 degrees C, an increase in the temperature promotes the spontaneous assembly into capsids of the Escherichia coli-expressed coat proteins of hepatitis B virus. Within that temperature interval, an increase in ionic strength up to five times that of standard physiological conditions also acts to promote capsid assembly. To explain both observations we propose an interaction of mean force between the protein subunits that is the sum of an attractive hydrophobic interaction, driving the self-assembly, and a repulsive electrostatic interaction, opposing the self-assembly. We find that the binding strength of the capsid subunits increases with temperature virtually independently of the ionic strength, and that, at fixed temperature, the binding strength increases with the square root of ionic strength. Both predictions are in quantitative agreement with experiment. We point out the similarities of capsid assembly in general and the micellization of surfactants. Finally we make plausible that electrostatic repulsion between the native core subunits of a large class of virus suppresses the formation in vivo of empty virus capsids, that is, without the presence of the charge-neutralizing nucleic acid.

Algorithms↗
PubMed2006

Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

Capsid↗
PubMed2005

Molecular characterization of the capsid gene of two serotypes of turkey astroviruses.

Astrovirus infections mainly cause acute gastroenteritis in children and young animals. Human astroviruses are well characterized antigenically and genetically. However, information on turkey astroviruses is limited. We isolated two astroviruses (TAstV1987 and TAstV2001) from turkeys and classified them as two different serotypes using a virus neutralization test. To elucidate the differences between these two isolates at the molecular level, further genetic characterization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were carried out. The sequences of the complete capsid protein gene of these two isolates were obtained by cloning and sequencing. The percentage nucleotide and predicted amino acid identities for these two sequences along with those of 16 other capsid protein gene sequences from human and animal astroviruses retrieved from GenBank were calculated using MegAlign. The results showed that TAstV1987 and TAstV2001 had 73.3% nucleotide and 82.8% amino acid identities, respectively. An unrooted Neighbor-joining phylogenetic tree of these sequences was generated using MEGA 3 software with 1000 bootstrap replicates. The results of evolutionary analysis showed that TAstV1987 was closely related genetically to another virus, designated TAstV-2, whereas TAstV2001 was not as close to TAstV-2 as TAstV1987. The analysis of the capsid proteins of the two viruses by SDS-PAGE revealed that they had different band patterns, indicating that their capsid proteins consisted of different viral proteins. The findings in this study revealed the molecular differences in the capsid protein gene of TAstV1987 and TAstV2001, which may provide the molecular basis of the antigenic differences between these two serotypes of turkey astroviruses.

Amino Acid Sequence↗
PubMed1983

Antigenicity and immunogenicity of poliovirus capsid proteins.

Poliovirus capsid proteins (VP's) were isolated by DEAE-Sepharose chromatography in the presence of RNA-se and 9 M urea or by preparative SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). With the first method, several, but not all, capsid proteins, from the three different virus types could be isolated in pure form, probably because of incomplete dissociation of the virus particles in 9 M urea. With SDS-PAGE all capsid proteins could be isolated in pure form. Immunization of rats and rabbits with the three largest VP's (VP1, VP2 and VP3) isolated by both methods, resulted in the induction of neutralizing antibodies already after 2-3 injections, provided the capsid proteins were isolated from formalin inactivated virus. Apparently formalin treatment stabilized the antigenic determinants, which are responsible for induction of neutralizing antibodies, against denaturation upon VP-isolation. In contrast to several other picornaviruses, where only one of the VP's induces neutralizing antibodies, for poliovirus both VP1, VP2 and VP3 are able to induce neutralizing antibodies. VP's isolated from live virus did not induce neutralizing antibodies, but were able to "prime" the animals. Primary vaccination with any of the VP's, followed by a second injection with a low dose of vaccine (D-antigen) resulted in a boosterlike production of neutralizing antibodies against the D-antigen, even D-antigen of a heterotypic virus. This indicates the occurrence of common antigenic regions on the different capsid polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

The isolation of Mengo virus stable non-capsid polypeptides from infected L cells and preliminary characterization of an RNA polymerase activity associated with polypeptide E.

Isolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS. Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.

Chromatography↗