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Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.

Animals↗

Anal cancer. Microscopic condyloma and tissue demonstration of human papillomavirus capsid antigen and viral DNA.

Sixteen cases of squamous cell carcinoma of the anus, including 4 incidentally discovered in situ lesions, and 3 anal condylomas, were examined for the presence of human papillomavirus (HPV). All in situ tumors and 6 of the invasive tumors were associated with histologic changes typical of condyloma, despite the absence of clinical anogenital warts. Immunohistochemical studies for viral capsid antigen gave positive reactions in two anal warts and in the condylomatous area associated with one invasive tumor. In situ hybridization was accomplished using isotopic DNA probes for HPV 6/11, 16, 18, and 31. Human papillomavirus 6/11 was expressed in the corresponding capsid-positive regions in the two warts and the wart-associated invasive carcinoma. Both HPV 6/11 and HPV 16 were associated with one carcinoma in situ, and HPV 16 was also found within two invasive anal carcinomas, one of which was associated with an extensive vulvar cancer. While these observations do not resolve the "passenger" or direct oncogenic role for HPV in anal carcinoma, the circumstantial evidence is that the oncogenic influence is similar to that accepted for female genital tract cancer.

Adult↗

[Preliminary results of the reactivity of T lymphocytes with HIV- capsid-antibodies].

HIV-infected and noninfected H9-cell cultures were incubated with two antibodies (Biochrom, DuPont) against p24gag antigen (capsid) of HIV: the antibodies show a positive reaction against p24 antigen in the Western blot. The antibody-labeled cells were examined by fluorescence-activated cell sorting (FACS) and by microscope. The labeled infected and uninfected cells show no characteristic fluorescence pattern after previous acetone fixation. By using formalinfixation technique the monoclonal antibody (DuPont) shows a positive fluorescence pattern but also a high unspecific reactivity with uninfected H9-cells. An identification of HIV-infected living cells with this monoclonal capsid antibody seems possible, if the unspecific binding can be reduced.

Antibodies, Viral↗

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA.

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.

Canavanine↗

Burkitt lymphoma. EBV nucleoid-like structures and lack of capsids in lymphoblasts.

Burkitt lymphoma biopsies and the CSF malignancy have been investigated ultrastructurally. The tumor cells were found to contain vermicellar bodies formed by rod-like structures similar to those suggested to represent HSV nucleoids in HSV encephalities. But, unlike the HSV disease and BL in culture, no nucleocapsids nor empty capsids were detected. It is suggested that the rods in the Burkitt lymphoblasts may well represent EBV nucleoids and that the absence of capsids may be another characteristic of the EBV in BL in situ.

Antigens, Viral↗

[Structure of the internal capsid of rotaviruses].

Several methodical approaches (negative staining, filtration by rotation, modeling, optic diffraction, and filtration of images) were used to study the structure of ther internal capsid of rotavirus and the structure and mutual arrangement of capsomers comprising it. The internal capsid of rotaviruses was shown to be constructed as a skew icosadeltahedron (left) with T-13 and to consist of 12 pentamers and 120 hexamers. The structural units (a total of 780) comprising capsomers are organized into trimers, each trimer participating in the formation of three adjacent capsomers, while each of the structural units is a part of only one capsomer.

Capsid↗

Induction of neutralizing antibodies by type 1 poliovirus VP1 capsid polypeptide.

Sera against purified VP1, VP2, and VP3 poliovirus capsid polypeptides of type 1 poliovirus were prepared in rabbits. Each serum was demonstrated to be specific for the polypeptide against which it was raised by immunoprecipitation with labeled, individualized capsid polypeptides. One of the anti-VP1 sera had a neutralizing capacity against type 1 poliovirus. It was the only one which reacted in immunoprecipitation with D-antigenic infectious particles.

Animals↗

Linking components between capsomers in the adenovirus capsid.

The interhexonal space within the adenovirus capsid is bridged by interhexonal connective elements. These linking components have been detected also between pentons and the five peripentonal hexons surrounding them. According to the results obtained by Markham's rotational integration technique the connective elements between the capsomers are interlacing the whole virion. It could be confirmed in several cases that the linking elements consisted of two approximately parallel elements. This means that each hexon is linked by six times two parallel linking components with its six nearest neighbours in the virus capsid.

Adenoviridae↗

Hybrid Rop-pIII proteins for the display of constrained peptides on filamentous phage capsids.

In order to increase the versatility of phage display technology, it is desirable to be able to impose some structural constraints on the peptides that are presented by the phage particles. This is currently not feasible since the conformation of the capsid proteins, used to link the foreign peptide to the phage, are either unknown (pIII) or too simple (pVIII) to permit the engineering of peptide inserts into a constrained context. To reach this scope we have modified the amino-terminus of gene III by appending a well-characterized protein motif, the four-helix bundle of the bacterial protein Rop. Phage particles displaying Rop can be separated from wild-type (wt) particles by affinity purification with an antibody. Rop can be extensively modified by substituting its solvent-exposed residues and/or by inserting peptides either into the carboxy-terminal tail or into the bend region that connects the two alpha-helices of the monomer. These results open the possibility to construct peptide libraries where the peptides are constrained either into an omega-loop type conformation or an alpha-helix. Libraries formed by peptides inserted into the carboxy-terminus can also be constructed. Furthermore, the system that we have developed permits to produce large quantities of the elements of the libraries in the cytoplasm or to display them on the capsid of filamentous phages.

Bacterial Proteins↗

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.

Amino Acid Sequence↗

Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: insights from computational and theoretical studies.

A network of protein vibrations has recently been identified in the enzyme cyclophilin A (CypA) that is associated with its peptidyl-prolyl cis/trans isomerization activity of small peptide substrates. It has been suggested that this network may have a role in promoting the catalytic step during the isomerization reaction. This work presents the results from the characterization of this network during the isomerization of the Gly89-Pro90 peptide bond in the N-terminal domain of the capsid protein (CA(N)) from human immunodeficiency virus type 1 (HIV-1), which is a naturally occurring, biologically relevant protein substrate for CypA. A variety of computational and theoretical studies are utilized to investigate the protein dynamics of the CypA-CA(N) complex, at multiple time scales, during the isomerization step. The results provide insights into the detailed mechanism of isomerization and confirm the presence of previously reported network of protein vibrations coupled to the reaction. Conserved CypA residues at the complex interface and at positions distal to the interface form parts of this network. There is HIV-1 related medical interest in CypA; incorporation of CypA, complexed with the capsid protein, into the virion is required for the infectious activity of HIV-1. Interaction energy and dynamical cross-correlation calculations are used for a detailed investigation of the protein-protein interactions in the CypA-CA(N) complex. The results show that CA(N) residues His87-Ala-Gly-Pro-Ile-Ala92 form the majority of the interactions with CypA residues. New protein-protein interactions distal to the active site (CypA Arg148-CA(N) Gln95 and CypA Arg148-CA(N) Asn121) are also identified.

Catalysis↗

Capsids of tricorn protease studied by electron cryomicroscopy.

Tricorn protease from the archaeon Thermoplasma acidophilum acts "downstream" of the proteasome; in conjunction with its aminopeptidase cofactors it converts peptides generated by the proteasome into free amino acids. The basic functional unit of Tricorn is a homohexamer of the 121-kDa subunit, 20 of which can assemble further to form an icosahedral capsid with a molecular mass of 14.6 MDa. We have used electron cryomicroscopy to determine the structure of the Tricorn capsids to a resolution of 1.3 nm.

Archaeal Proteins↗

Interaction of the capsid protein p24 (HIV-1) with sequence-derived peptides: influence on p24 dimerization.

Protein-protein interactions of the p24 (HIV-1) capsid protein play an essential role in the production of infectious virus particles. To map the putative p24 dimerization site, a set of overlapping peptides spanning the p24 sequence was prepared using spot synthesis on a cellulose membrane and probed with recombinant p24 (rp24). Three sequence regions interacting with rp24 were identified. Peptides from each region were synthesized, but only one peptide was effectively able to inhibit rp24 dimerization in solution. Amino acids that were exposed in the corresponding p24 region were mutated in rp24, resulting in a significant decrease of rp24 dimerization. Thus, participation of this region in virus capsid assembly can be assumed.

Animals↗

Detection of enterovirus capsid protein VP1 in myocardium from cases of myocarditis or dilated cardiomyopathy by immunohistochemistry: further evidence of enterovirus persistence in myocytes.

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected chi(2)=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.

Animals↗

Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures.

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.

Animals↗

A recombinant human immunodeficiency virus type-1 capsid protein (rp24): its expression, purification and physico-chemical characterization.

An expression system has been established in Escherichia coli to facilitate the preparation of the HIV-1 capsid protein in amounts sufficient for structural analysis. A plasmid vector pTCA5, containing the gene for the recombinant HIV-1 capsid protein rp24 under the control of the lambda-PR-promoter, was constructed which gave an expression product that spanned 234 amino acid residues. It differs at the N-terminus from the authentic sequence in that the residues Pro-Ile- are replaced by Met-Asn-Ser-Ala-Met-. Recombinant p24 was produced, as inclusion bodies in E. coli LE392 containing pTCA5, at a level of approximately 15% of the total cellular protein. After dissolution of the inclusion bodies in the acidic urea system, the protein was easily reconstituted in a soluble state by dialysis. The yield of reconstituted and purified protein was 12 mg per liter in rich medium. Recombinant rp24 consists of about 40% alpha-helix and 10% beta-sheet from circular dichroism measurements and the two cysteine residues, within the rp24 sequence, are bridged by a disulfide bond.

Base Sequence↗

Isolation and characterization of an arildone-resistant poliovirus 2 mutant with an altered capsid protein VP1.

Arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl] -3,5-heptanedione is a selective inhibitor of poliovirus uncoating. We have isolated an arildone-resistant poliovirus 2 variant and characterized it. In single cycle growth curve experiments the resistant virus was not influenced by 1 microgram/ml of arildone, which completely inhibited the replication of the sensitive virus. In neutralization experiments the rates of inactivation vs time of the parent and the resistant virus proved indistinguishable. The adsorption behavior of the mutant closely resembled that of the wild-type. Uncoating as measured with neutral red-sensitized arildone-resistant mutant virus was unaffected by arildone, in contrast to the uncoating of the sensitive parent virus. It has been previously shown that arildone stabilizes the poliovirus capsid from alkaline degradation. The arildone-resistant mutant, however, was not markedly stabilized by the drug under these conditions. No difference in thermostability between mutant and parent virus could be detected. In isoelectric focusing the sensitive virus exhibited 2 peaks, but in the presence of arildone only one peak was apparent. In contrast, the arildone-resistant virus remained unaffected by the presence of arildone. Analysis of the capsid proteins of the sensitive parent and the resistant virus in SDS-polyacrylamide gels revealed no difference in the patterns; however, peptide mapping showed clear differences in VP1.

Animals↗

SDZ 35-682, a new picornavirus capsid-binding agent with potent antiviral activity.

SDZ 35-682 is a potent and selective inhibitor of the replication of members of the picornavirus group. It inhibits several rhinovirus serotypes and echovirus 9 at concentrations as low as 0.1 micrograms/ml, without exerting any effect on cell proliferation up to 30 micrograms/ml. As observed with other capsid-binding antipicornavirus compounds, there is a wide variation in sensitivity of the different serotypes within the rhinovirus group. The point of interference of SDZ 35-682 in a single cycle of virus growth is an early event taking place before 2 or 3 h of echo- or rhinovirus replication, respectively. By incorporation of neutral red into the viral capsid and measurement of acquisition of photoresistance it is shown that uncoating of echovirus 9 is inhibited by SDZ 35-682. In addition, efficiency of adsorption of echovirus 9 is reduced by SDZ 35-682. To demonstrate that SDZ 35-682, like other uncoating inhibitors of picornaviruses, binds to the hydrophobic pocket beneath the canyon floor co-crystallization with HRV 14 was performed. Considerable conformational changes occur in VP1 in the HRV 14/SDZ 35-682 complex. SDZ 35-682 is 19 A long from end to end and thus fills the entire hydrophobic pocket including its innermost end; it is less flexible than other long antiviral agents. It has been suggested that compounds filling the entire hydrophobic pocket will affect the uncoating process of the virion. Thus, inhibition of viral uncoating, as demonstrated with echovirus 9, probably is the predominant mode of action of SDZ 35-682.

Animals↗