A re-appraisal of the biochemical map of foot-and-mouth disease virus RNA.

The proteins induced by infection of BHK 21 cells with foot-and-mouth disease virus have been compared by tryptic peptide analysis. The results indicate that there are three primary products 5'--P88, P52, P100--3'. The polypeptide P56, which we considered previously to be a primary product, is derived from the region of the genome that codes for P100. The results indicate that there are alternative cleavage pathways of P100, the polypeptide coded for by the 3' end of the genome.     

Biochemical aspects of variation in foot-and-mouth disease virus

The biochemical basis for variation in foot-and-mouth disease virus (FMDV) has been explored by analysis of the virus RNA and the virus-induced and structural proteins of three isolates of the virus. Two of the isolates were from serotype A and the third was from serotype O. Hybridization studies of the RNAs showed greater than 80% homology between the two type A viruses and about 65% homology between the two type A viruses and the virus of type O. The ribonuclease T1 maps of the three viruses gave distinct patterns typical of FMDV, but did not show that any two of the three viruses were more closely related. The virus-induced primary translation products, P88, P52 and P100 isolated from infected cells, were compared by tryptic peptide analysis. Combinations of 3H- and 14C-leucine-labelled polypeptides were hydrolysed with trypsin and resolved on an ion-exchange column. Much greater differences were found in P88 than in P52 or P100, indicating that the major variation occurs in the region of the genome coding for the structural proteins. Similar analysis of combinations of the structural proteins of the three viruses showed that there were differences in VP1, VP2 and VP3 and these results were supported by those obtained by PAGE analysis of the Staphylococcus aureus V8 protease cleavage products.     

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped + ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.     

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.     

Cytoskeletal association of an aphthovirus-induced polypeptide derived from the P3ABC region of the viral polyprotein

Monolayers of BHK cells infected with aphthovirus (FMDV) were labeled for short times with [35S]methionine at 2 hr p.i. and fractionated by detergent treatment and low speed centrifugation. Polyacrylamide gel analysis showed an asymmetric distribution of the FMDV-induced polypeptides among the three different subcellular fractions obtained. Polypeptide P88-1, the viral capsid protein precursor, is mainly found in the soluble cytoplasmic extract while polypeptides P100-3, P52-2AC, P34-2C, and P14-2A are the major viral components of a detergent soluble extract of the crude nuclear pellet. Analysis of the detergent resistant fraction (DRF), which is mainly composed of cell nuclear chromatin and insoluble cytoskeletal elements, shows a clear enrichment in an incompletely characterized polypeptide which is tentatively designated P54. Variable amounts of polypeptides P100-3 and those of the P72 complex are also detected in this fraction. The preferential location of P54 in an equivalent subcellular fraction obtained by mild detergent treatment of infected monolayers in situ, and also in a high-salt resistant subfraction of the DRF, strongly suggests a close association of this polypeptide with vimentin-actin containing components of the cell. Polypeptide P54 is immunoprecipitated by viral specific antiserum from convalescent guinea pigs but not by serum against FMDV capsid proteins, indicating that it does not share common antigenic determinants with polypeptides processed from the viral capsid precursors. On the other hand, protease V8 mapping of polypeptides P100-3, P54, P88-1, and VP1-3 shows that P54 derived from the 3' end coding region of the viral genome. Further analysis by limited protease digestion also demonstrates that P54 has partial overlap with P72-3CD while it does not share any common peptide with P56a-3D, indicating that P54 contains the sequences coded in the 3ABC region of the FMDV RNA. This assumption is reinforced by the basic behavior shown by P54 in two-dimensional gels. The results support the hypothesis of a close intracellular interaction of a short-lived polypeptide, containing the viral protease and VPg sequences, with the host cytoskeleton, during infection of BHK cells with FMDV.     

Synthesis of Drosophila X virus proteins in cultured Drosophila cells

The genome of Drosophila X virus (DXV) is made up of two segments of dsRNA with molecular weights of 2.3 X 10(6) (A) and 2.2 X 10(6) (B). Agarose gel electrophoresis of RNA fragments produced by S1 nuclease digestion of partially denatured, purified segment A and B indicated that the two genome segments have different nucleotide sequences. The dose-response curve of virus infectivity was linear, indicating that the two genome segments reside in the same particle. The virions contained five polypeptides (VPs) that fell into three molecular weight size classes; large, 110K; medium, 49K and 45K; and small, 34K and 27K. Virus infection of Drosophila cells induced the synthesis of five infected cell polypeptides (ICPs); 110K, 67K, 49K, 34K, and 27K. Pulse-chase experiments and peptide mapping revealed that four of these (110K, 67K, 34K, and 27K) were primary gene products and that ICP 49 was generated by post-translational cleavage of ICP 67. The major capsid protein, VP 45, was cleaved from ICP 49; however, this cleavage was incomplete because both polypeptides were present in purified virus. The results suggest that the strategy for protein synthesis of DXV differs from that of other dsRNA viruses.     

Study of a new strain of paramyxoviruses isolated from wild ducks: structural polypeptides.

By polyacrylamide gel electrophoresis, Duck/Mississippi/75 virus was shown to contain five different types of polypeptides of mol. wt. ranging from 76 X 1O(3) to 43 X 10(3), two of which were glycosylated (mol. wt. 76 X 10(3) and 57 X 10(3). A comparison of this data with similar results obtained using Yucaipa and Newcastle disease virus (NDV) revealed a similarity with NDV, concerning the number, position and mol. wt. of the polypeptides. Haemagglutinating and neuraminidase properties are associated with surface glycoproteins which represent at least 40% of the virus protein. After KCl and Triton X-100 treatment and centrifugation on linear sucrose density gradients (10 to 25%, w/w) it was possible to isolate glycoprotein VP1 of mol. wt 76 X 10(3) with which haemagglutinating and neuraminidase activities were associated.     

Identification of the simian rotavirus SA11 genome segment 3 product

Previous studies on rotavirus gene-coding assignments failed to clearly identify the simian rotavirus SA11 genome segment 3 protein product. This question was reexamined by using new conditions of electrophoresis with improved resolution of proteins in the high-molecular-weight range. Our results showed that the SA11 genome segment 3 codes for a protein with an apparent mol wt of 88,000. This protein normally comigrates with the protein product of genome segment 4. The gene 3 protein was located in viral core particles by comparing the electrophoretic patterns of [35S]methionine-labeled viral polypeptides from infected cells and from purified double-shelled, single-shelled, and core particles. To confirm the identity of the gene 3 product, we also studied two reassortant viruses in which genome segment 3 was reassorted from each of two parental virus strains (SA11 and NCDV). The gene 3 and gene 4 products of these viruses were identified by (i) their separation by two different polyacrylamide gel systems, (ii) their location in distinct particle types, (iii) their differential sensitivity to trypsin digestion, and (iv) their distinctive protease peptide maps. We propose that the genome segment 3 product be called VP3 and that the gene 4 product be named VP4 from now on.     

Effect of trypsin and chymotrypsin on the polypeptides of large and small plaque variants of foot-and-mouth disease virus: relationship to specific antigenicity and infectivity.

Large and small plaque variants of A12 foot-and-mouth disease virus were shown to have specific antigenic determinants. Large plaque virus antigenic specificity was destroyed by trypsin treatment, but the small plaque antigen was resistant despite cleavage of the trypsin-sensitive polypeptide. The cleavage of polypeptide VP3 by trypsin resulted in the formation of a new antigen not present on untreated virus. The effects of chymotrypsin and trypsin on the polypeptides of the plaque variants have been examined and related to changes in antigenicity, infectivity, and exposure of the polypeptides at the surface of the capsid. The results are discussed in relation to the orientation of the trypsin-sensitive polypeptide in the virus capsid.     

The immunoglobulin response to individual HSV-1 viral polypeptides: kinetics of the response during primary and secondary experimental infection with herpes simplex virus

We studied the immunoglobulin response to individual viral polypeptides in experimental primary and secondary infection with herpes simplex virus (HSV)-1 in mice. After a single footpad inoculation with 10(5.6) pfu of HSV-1, immunoglobulin to proteins mol. wts (10(3)) 44 and 75 appeared on day 7. Antibodies to gB, gC, gD, 42 x 10(3)- and (48-52) x 10(3)-mol. wt proteins appeared on day 11 and antibody to the major capsid protein, VP154, appeared on day 15 after infection. The secondary immune response was characterised by early production of antibody to gD on day 3 followed by antibodies against the 42 x 10(3)- and 44 x 10(3)-mol. wt proteins on days 4 and 5 respectively. Antibodies to glycoprotein gC and gB were delayed until day 7 of the secondary immune response. In both primary and secondary immune responses the responses against proteins of mol. wts (10(3)) 42 and 44 were particularly intense and of high titre. We conclude that the kinetics of anti-polypeptide antibody appearance is markedly asynchronous; and that the anti-glycoprotein responses occur too late in primary infection to contribute to viral clearance.     

Poliovirus polypeptides examined in more detail.

The pattern of viral polypeptide synthesis and cleavage in poliovirus-infected cells was shown by autoradiography to be considerably more complex than previously thought. In normal growth, at least 26 distinct polypeptides were found, and various modifications of the cleavage process revealed a total of at least 34. Most of the new polypeptides were minor components that were unstable during a chase. Different cultural modifications led to different polypeptide ratios, and it appeared likely that several cleavage activities were involved . Minor differences were found in the polypeptide contents of cytoplasmic extracts and whole infected cells. The complexity of the cleavage pattern necessitated a new nomenclature based on mol. wt. (e.g. Pp110, "poliovirus protein" of 110000). Particular attention was paid to mol. wt. determinations, notably in the use of internal protein standards and more fully denaturing gel conditions. The size of the "primary translation product" of poliovirus RNA was found to be 210000 daltons, so that either 20% of the viral genome is not translated in vivo, or some is read as a smaller independent translation unit.     

Immunogenic and cell attachment sites of FMDV: further evidence for their location in a single capsid polypeptide.

Chymotrypsin cleaves only one of the four major polypeptides of foot-and-mouth disease virus (FMDV serotype O) in situ. This polypeptide (VP1, mol. wt. 29 X 10(3) was first cleaved into fragments of mol. wt. 20 and 9 X 10(3) and further cleavage could be prevented by the addition of a large excess of bovine serum albumin. The infectivity of the virus particles at this stage was the same as that of the intact virus although the rate of attachment to BHK 21 cells was slower and the immunogenic activity was reduced. If hydrolysis was allowed to continue, VP1 was cleaved into fragments with mol. wt. 18 and less than 9 X 10(3), similar to those obtained with trypsin and the virus particles then had a greatly reduced infectivity and a lower immunogenicity. Treatment of strains from five other serotypes of the virus with the two enzymes cleaved only VP1 in each instance and there was a corresponding loss of infectivity. The results are discussed in relation to the location and biological activity of the virus polypeptides.     

Coding assignments of Drosophila X virus genome segments: in vitro translation of native and denatured virion dsRNA

Both dsRNA genome segments of Drosophila X virus (DXV) were denatured and translated in vitro using nuclease-treated rabbit reticulocyte lysates. The synthesis of all four primary gene products was detected by polyacrylamide gel electrophoresis and autoradiography. Genome segment A (mol wt 2.3 X 10(6)) encoded polypeptides with molecular weights of 67,000 (67K), 34K, and 27K, whereas segment B (mol wt 22 X 10(6)) encoded the 110K polypeptide. The proteolytic processing of 67K which generates a 49K polypeptide in infected cells was also observed in vitro. Pulse-chase experiments indicated that synthesis of the three polypeptides encoded by genome segment A initiated independently and simultaneously, suggesting that segment A is polycistronic. Native (undenatured) DXV dsRNA could also be translated with high fidelity (vitro). The messenger activity of native dsRNA was abolished by S1 nuclease treatment but completely restored on subsequent denaturation. In vitro "pulse-chase" experiments using native dsRNA as messenger, indicated that the order of translation of polypeptides on genome segment A was 5'-67K-27K-34K-3'.     

Topographical studies on poliovirus capsid proteins by chemical modification and cross-linking with bifunctional reagents.

Poliovirus capsid proteins comprise 15.1 lysines in VP1, 5.6 lysines in VP2, 11.7 lysines in VP3 and 5.5 lysines in VP4. Treatment with monofunctional reagent N-succinimidyl 2,3-3H-proprionate leads to the modification of 3.4 lysines in VP1, 0.6 lysines in VP2, 2.0 lysines in VP3 and 0.03 lysines in VP4. Chemical modification with the monofunctional reagent N-succinimidyl 3-(4-hydroxy,5-125I-iodophenyl)propionate results in a predominant labelling of VP1 and VP3, whereas VP2 is less accessible and VP4 is not modified. Cross-linking of poliovirus with bifunctional imidoesters, dimethyl suberimidate (DMS, 1.1 nm) and dimethyl adipimidate (DMA, 0.8 nm) leads to a new protein complex of mol. wt. which corresponds to the sum of VP1 and VP3. By cleavage with ammonia and electrophoresis on polyacrylamide gels in SDS, the proteins are identified as VP1 and VP3. This result gives evidence for a direct neighbourhood of VP1 and VP3 in the virus capsid. Treatment of the virus with the mono- and bifunctional reagents has no influence on the stability of the particle. The infectivity is reduced only by the bifunctional reagent.     

Relations between poliovirus polypeptides as shown by tryptic peptide analysis.

Poliovirus proteins were labelled in vivo with [35S]-methionine, and the major products of translation and cleavage were separated by electrophoresis and compared in terms of two-dimensional tryptic peptide maps visualized by autoradiography. The main intermediates p110 and p90 had few or no methionine-labelled sequences in common, but were both contained in, and therefore almost fully account for, the presumed primary translation product p210. The sequences of p79, a major stable product of cleavage and a non-structural protein, were almost completely contained in p90, which in turn is the major component of the larger intermediates p168 and p155. P110 is confirmed as the precursor of virus particle protein, and VP0 as the precursor of VP2. However, the sequences of p31, the other major product of translation that is not a stuctural protein, were not contained in any of the viral polypeptides mentioned above.     

Synthesis of viral-specific polypeptides in Mengo virus-infected L cells: evidence for asymmetric translation of the viral genome

Investigations of the synthesis of Mengo virus-specific polypeptides in L cells during the logarithmic phase of virus production have shown that, as is the case with other picornaviruses, the stable viral gene products are formed by posttranslational cleavage of larger primary proteins and that the pattern of cleavages by which they are produced is very similar to that proposed earlier for EMC virus (Butterworth et al., 1972b). However, analysis of the data suggests strongly that, on a molar basis, twice as much capsid as noncapsid viral protein is synthesized. This, in turn, suggests that translation of the viral genome is under some kind of control, the model most compatible with the data being one in which some viral protein(s) functions as termination factor(s).     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).     

Immune response to hepatitis A virus capsid proteins after infection.

This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.     

Proteins induced by infection with caliciviruses.

Three polypeptides with mol. wt. 100 (P100), 80 (P80) and 65 (P65) X 10(3) were found in calicivirus infected cells. P100 and P80 were present in sub-molar amounts compared with P65 and no precursor product relationship between the three polypeptides could be demonstrated using pulse-chase experiments or selective inhibitors of protein synthesis and of proteases. In the presence of protease inhibitors a polypeptide with mol. wt. 120 X 10(3) (P120) was demonstrated which appeared to be the precursor of P100. Possible mechanisms of translation in the caliciviruses are discussed.