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PubMed1983

Induction of neutralizing antibodies by poliovirus capsid polypeptides VP1, VP2 and VP3.

The antigenic and immunogenic properties of poliovirus capsid polypeptides (VPs) have been investigated in vivo and in vitro. VPs were isolated from infectious and formalin inactivated virus. Upon immunization VPs from infectious virus did not induce neutralizing antibodies in laboratory animals after two or three injections. Monomeric VP1, VP2 and VP3 from formalin inactivated virus all induced high titres of neutralizing antibodies generally already after two injections. Some anti-VP sera neutralized heterologous virus types. All anti-VP sera reacted with the corresponding capsid polypeptides from the homologous and heterologous virus strains in an immunoblotting assay. The anti-VP sera reacted equally well with D- and C-antigen in ELISA. Our results indicate that not only VP1, but also VP2 and VP3 are involved in the induction of neutralizing antibodies.

Animals↗
PubMed2026

Alternative bipartite arrangements of VP1 BR1-3 drive efficient nuclear import of AAV2 capsids.

Adeno-associated viruses (AAVs) are among the most extensively studied viral gene-therapy vectors, yet the mechanisms governing their nuclear entry remain incompletely understood. Efficient transduction requires that the AAV capsid, or its structural subunit VP1, traverse the nuclear envelope to deliver the therapeutic genome. The N-terminal region of VP1 contains three clustered basic regions (BR1-3) proposed to function as nuclear localization signals (NLSs). Here, we combine cellular, biophysical, structural, and computational modelling approaches to define the nuclear import mechanism of AAV2 VP1 at molecular resolution. We show that VP1 engages the classical importin-α/β1 (IMPα/β1) pathway and binds multiple IMPα paralogs with distinct affinities. Crystallographic and mutational analyses reveal that two intact BRs are required to simultaneously occupy the major and minor binding pockets of IMPα in a bipartite configuration. Structural data indicate that mouse IMPα2 (mIMPα2) preferentially accommodates BR1 and BR3 at these sites, however, functional studies demonstrate that mutation of individual BRs does not abolish IMP binding or nuclear accumulation. This robustness arises from the ability of BR2 to flexibly engage both binding pockets, enabling the formation of alternative bipartite arrangements (BR1-BR2, BR2-BR3, or BR1-BR3). Together, these findings reveal an unexpected versatility in how AAV2 VP1 exploits the IMPα binding sites, providing a structural basis for efficient capsid nuclear import. The flexibility of BR1-3 expands the current paradigm of viral NLS organization and suggests new strategies to fine-tune nuclear targeting AAV-based gene-therapy vectors.

Adeno-associated virus↗
PubMed2005

High-level expression and immunogenic properties of the recombinant rabbit hemorrhagic disease virus VP60 capsid protein obtained in Pichia pastoris.

The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.

Animals↗
PubMed2004

The C-terminal domain of the pVP2 precursor is essential for the interaction between VP2 and VP3, the capsid polypeptides of infectious bursal disease virus.

The interaction between the infectious bursal disease virus (IBDV) capsid proteins VP2 and VP3 has been analyzed in vivo using baculovirus expression vectors. Data presented here demonstrate that the 71-amino acid C-terminal-specific domain of pVP2, the VP2 precursor, is essential for the establishment of the VP2-VP3 interaction. Additionally, we show that coexpression of the pVP2 and VP3 polypeptides from independent genes results in the assembly of virus-like particles (VLPs). This observation demonstrates that these two polypeptides contain the minimal information required for capsid assembly, and that this process does not require the presence of the precursor polyprotein.

Baculoviridae↗
PubMed1999

Recombinant, attenuated Salmonella typhimurium stimulate lymphoproliferative responses to SIV capsid antigen in rhesus macaques.

Recombinant bacteria are useful vectors for delivering foreign antigens to mucosal surfaces and may elicit immune protection against sexually-transmitted pathogens. Recombinant, attenuated Salmonella typhimurium expressing the Simian Immunodeficiency Virus capsid protein (p27) were given to rhesus macaques by intragastric intubation. This route of immunization was compared with intramuscular injection of soluble p27 in adjuvant, and with immunization protocols that combined intragastric and intramuscular antigen exposures. Recombinant Salmonella stimulated p27-specific lymphoproliferative responses that were present transiently in peripheral blood, and were recalled easily by booster immunizations. Intramuscular p27 injection elicited strong serum antibody responses, but only low level capsid-specific proliferative responses. Recombinant Salmonella immunization elicited low levels of p27-specific antibodies in serum and did not suppress subsequent responses to parenteral immunization. Intragastric immunization of macaques with recombinant Salmonella typhimurium was safe and induced immune responses specific for the expressed, foreign antigen.

Animals↗
PubMed1974

Cell-free translation of messenger RNA of simian virus 40: synthesis of the major capsid protein.

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.

Adenine Nucleotides↗
PubMed1975

Quasi-elastic behavior of solutions of viral capsid and RNA at very low shearing stresses.

By the application of shearing stresses on the order of 10(-3) dyne cm-2 (10(-2) muN cm-2), via the magnetic viscodensimeter, extremely high relative viscosities (greater than 500) were observed when turnip yellow mosaic virus was degraded in alkali into its capsid and RNA. The solutions, however, possessed a watery consistency at this stage and exhibited a quasi-elastic character by rotor-recoil experiments. The development of this curious behavior was concentration and temperature dependent; it was not seen less than 0.5% nor at 8 degrees, and appeared sooner at 30 degrees than at 20 degrees. The time of appearance was delayed as the pH was lowered; however, the effect was still observed when the pH was as low as 9. Whereas reversibility was demonstrated when the shearing stresses exceeded the elastic resistance [0.17 dyne cm-2 (1.7 muN CM-2)], thorough mixing usually resulted in a normal behavior of the solutions thereafter. Values for the modulus of rigidity at 20 degrees for about 1% virus concentration was less than 2 X 10(-2) dyne cm-2 rad-1 (0.2 muN cm-2 rad-1), which, while extremely small, was reproducible. A porous structure, possibly involving a capsid and RNA complex, is envisioned.

Mosaic Viruses↗
PubMed1978

DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes.

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.

Animals↗
PubMed1979

Ultrasonic absorption evidence of structural fluctuations in viral capsids.

When the coat protein of the small icosahedral virus, brome mosaic virus, reassembles into capsids, the ultrasonic absorption of the solution greatly increases. Submitting the solution to an ultrasonic field thus appears to reveal spontaneous molecular motions within a protein assembly. Confirmatory evidence of a dynamics of a protein shell comes from measurements on brome mosaic virus at various degrees of swelling and on tomato bushy stunt virus treated with the crosslinking agent glutaraldehyde. The detected fluctuations may be related either with cooperative deformational motion in the capsid or with more localized structural changes. Such structural changes may help liberate the RNA at an early stage of viral infection.

Mosaic Viruses↗
PubMed1985

Sequence analysis of hepatitis A virus cDNA coding for capsid proteins and RNA polymerase.

We report here the nucleotide sequence corresponding to two large regions of the hepatitis A virus (HAV) genome. These comprise a sequence of 3274 bases corresponding to the 5' end of the genome, which includes the putative capsid protein region of this picornavirus, and 1590 bases corresponding to the 3' end of the genome, terminating in a 15-base poly(A) tract. These sequences revealed that HAV had the characteristic genomic organization of picornaviruses: an open reading frame beginning approximately 750 bases from the 5' end of the RNA and a termination codon 60 bases from the 3' poly(A) tract. The predicted amino acid sequences of both regions have been compared to analogous regions previously determined for other picornaviruses. There was sufficient homology to conclude that the 5' region of HAV codes for capsid proteins and that the 3' region codes for an RNA polymerase. However, these regions of HAV were not found to be closely related to analogous regions of poliovirus, encephalomyocarditis virus, and foot and mouth disease virus.

Amino Acid Sequence↗
PubMed1988

A point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in Nicotiana sylvestris.

In Nicotiana sylvestris, the N' gene confers hypersensitive resistance to some strains of tobacco mosaic virus (TMV) but not to the common strain. TMV sequences responsible for inducing local lesion formation in this host were identified by using cDNA clones to construct genomic recombinants between the common strain genome and a local-lesion-inducing mutant. To assay for sequences conferring the mutant phenotype, in vitro transcripts of recombinants were inoculated onto leaves of N. sylvestris and observed for the formation of either local lesions or a systemic infection. Sequences from the mutant that converted the hybrid genome to the mutant phenotype were located between nucleotides 5972 and 6206. Sequence analysis of this region revealed point mutations in the mutant at nucleotides 6157 (cytosine to uracil) and 6199 (adenine to guanine). The mutation at 6157 changes the capsid protein gene to specify phenylalanine rather than serine at position 148; nucleotide 6199 occurs in the 3' nontranslated region. When each point mutation was individually substituted into the wild-type background, transcripts containing only the alteration at 6157 produced local lesions on N. sylvestris, whereas transcripts containing only the alteration at 6199 produced systemic mosaic symptoms. The frequency of mutation was examined by partially sequencing virion RNA from six additional independent local-lesion mutants. Five mutants had the same alteration at 6157 as the original mutant and none had the alteration at 6199. This work demonstrates that the capsid protein gene of TMV is multifunctional, both encoding the virion structural protein and mediating the outcome of infection in N. sylvestris.

Journal Article↗
PubMed1992

Development of a monoclonal antibody-based p24 capsid antigen detection assay for HTLV-I, HTLV-II, and STLV-I infection.

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and employed to detect p24 capsid antigen from human T-cell lymphotropic viruses type I and II (HTLV-I, HTLV-II), simian T-cell lymphotropic virus type I (STLV-I)-infected cell lines, and from mononuclear cell cocultures of HTLV-infected humans and STLV-I infected monkeys. A monoclonal antibody specific for HTLV p24 and p53 capsid antigens was coated onto 96-well microtiter plates to capture HTLV/STLV antigen. Captured antigen was then detected by the addition of a polyclonal, biotinylated human anti-HTLV-I antibody, and color developed with tetramethyl benzidine/H2O2 substrate. As little as 15 pg/ml of HTLV-I p24 antigen could be detected in this assay. Culture supernatants from HTLV-I-infected cell lines (HUT-102, MT-2, C5/MJ, HTLV-II-infected cell lines (Mo-T, Mo-B, PanG 12.1, NRA) and STLV-I-infected cell lines (Matsu, NEPC M39) were all positive in the assay. In addition, p24 was detected from peripheral blood mononuclear cell (PBMC) cocultures of 8 of 8 (100%) HTLV-I diseased patients, 14 of 20 (70%) HTLV-I and HTLV-II-infected, asymptomatic persons, and 8 of 8 (100%) STLV-I-infected, asymptomatic monkeys. Culture supernatants of cells infected with human immunodeficiency virus type (HIV-1), simian immunodeficiency virus (SIV), Chlamydia trachomatis, cytomegalovirus (CMV), herpes simplex I and II (HSV), feline leukemia virus (FELV), bovine leukemia virus (BLV), and bovine immunodeficiency virus (BIV) were all negative. Similarly, normal human peripheral blood mononuclear cells and uninfected, transformed human T cells, were also negative in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed2002

Fate of plasmid DNA encoding infectious bursal disease virus VP2 capsid protein gene after injection into the pectoralis muscle of the chicken.

The objective of this study was to determine whether recombinant plasmid DNA injected intramuscularly into chickens expressed the gene of interest in vivo and could be subsequently detected in primary and secondary lymphoid tissues with polymerase chain reaction (PCR). The VP2 capsid protein gene of the standard challenge strain (STC) of infectious bursal disease virus (IBDV) was cloned into a eukaryotic plasmid, and purified DNA was prepared. Fourteen 2-wk-old chickens were injected in the pectoral musculature with 500 microg of plasmid DNA dissolved in sterile PBS. Seven chickens were similarly injected with PBS alone. Pectoral muscle, thymus, spleen, bursa of Fabricius, and cecal tonsils were collected at 12, 24, 36, 48, 72, 96, and 168 h postinjection for detection of protein expression (in muscle) and to extract total DNA for PCR amplification of the VP2 capsid gene. Expression of VP2 was demonstrated in muscle tissue at 12 and 24 h postinjection by using an indirect immunofluorescence assay. PCR amplification with primers specific for the VP2 gene showed that the DNA was present in the thymus, spleen, and bursa of Fabricius but not in cecal tonsils. These results demonstrate that plasmid DNA injected directly into the pectoral muscle of chickens is transcribed and translated at the injection site and promptly distributed to primary and secondary lymphoid tissues.

Animals↗
PubMed1979

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.

Chemical Phenomena↗
PubMed2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We employed protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a supefamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains, and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

adenovirus↗
PubMed1995

WIN51711-resistant mutants of poliovirus type 3: capsid residues important in uncoating functions.

Capsid-binding drugs that inhibit the first stage of picornaviral uncoating were used to select drug-resistant mutants of the Sabin strain of poliovirus type 3. Such mutants provide information about parts of the capsid that are important for functions blocked by the drugs, and also about pathways to drug resistance. Amino-acid substitutions allowing virus to produce progeny in the presence of drug were mapped to 13 different residues occupying three distinct locations: (I) the canyon base; (II) the lining of the drug-binding pocket; and (III) the base of the protomer. These loci might be thought of as action points for transmitting the uncoating signal from receptor, through the pocket, and to the base of the protomer. All of the mutations in a special class of drug-dependent mutants were clustered at site (III) and all were hyperlabile, i.e., uncoated spontaneously (without receptor) at growth temperature unless prevented from doing so by the presence of drug in the pocket. Thus, site (III) seems to represent a kind of thermostat which regulates the temperature at which the uncoating transition (release of VP4 to form A particles) is triggered.

Journal Article↗
PubMed1993

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk.

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.

Journal Article↗
PubMed1974

Reactivity of envelope, capsid, and soluble antigens of Herpesvirus hominis types 1 and 2 in the indirect hemagglutination test.

Envelope, capsid, and soluble antigens of Herpesvirus hominis (HVH) types 1 and 2 were compared to crude antigens (disrupted HVH-infected cells) for potency, type-specificity, and diagnostic value in the indirect hemagglutination (IHA) test. The envelope appeared to be the predominant component reacting in the IHA test, but recurrent HVH infections increased the reactivity of human sera with capsid antigens. The soluble antigens reacted in the IHA test with HVH immune animal sera, but very few convalescent-phase human sera showed reactivity with soluble antigens. Overall, none of the subunit antigens showed greater type-specificity than did crude antigens in IHA tests with immune animal or convalescent-phase human sera. Recurrent infections with type 1 or type 2 viruses tended to broaden heterotypic reactivity of the patients' sera with both crude and subunit antigens, even in patients showing only a single type of antibody by IHA inhibition. Subunit antigens were no more sensitive than crude antigens in demonstrating significant IHA antibody titer rises for serodiagnosis of herpesvirus infections, and they generally had to be used at lower working dilutions than crude antigens.

Animals↗