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At least 289 records · Page 16Linked to original sources
PubMed1988

Primate cytomegalovirus assembly: evidence that DNA packaging occurs subsequent to B capsid assembly.

Results presented here show that when cytomegalovirus (strain Colburn)-infected cells are treated with the DNA synthesis inhibitor hydroxyurea or phosphonoformate, one type of intranuclear capsid accumulates. These particles appeared to contain symmetrically organized internal material, and had a protein composition and sedimentation rate characteristic of B capsids. Radiolabeling experiments provided evidence that a population of B capsids lacking DNA is present during the course of a normal infection. These capsids sedimented slightly slower than the peak of viral DNA in the same region of the gradient, and had a ratio of DNA/protein that was estimated to be sevenfold lower than that of the faster sedimenting C capsids. DNA in both the B and C capsid regions of such gradients was found to be relatively resistant to digestion with DNase. The possibility is considered that herpesvirus B capsids lacking DNA may be counterparts of unexpanded proheads in the bacteriophage assembly pathway.

Capsid↗
PubMed1994

Analysis of capsid protein gene variation among divergent isolates of feline calicivirus.

Genomic variability within the capsid protein gene of feline calicivirus (FCV) was evaluated among different isolates using hybridization analysis and enzymatic viral nucleic acid amplification. Total infected cell RNA was first hybridized with cDNA clones generated to the capsid gene of the FCV isolates CFI/68, 255, LLK, NADC and KCD. Field isolates of FCV were categorized by hybridization with capsid gene cDNA from the reference strains. Isolates that did not hybridize were positive by Western blot using a cross-reactive cat polyclonal FCV CFI/68 capsid protein antiserum. Using previously published sequence information, oligonucleotide primers were generated based on conserved sequences surrounding the hypervariable capsid protein gene regions. Analysis of the FCV capsid protein gene hypervariable regions was completed by sequencing products of FCV nucleic acid amplified by reverse transcription and polymerase chain reaction. From these data, amino acid substitutions in the hypervariable regions of the capsid protein were identified for those isolates that did not hybridize with the original cDNA clones. An association between phylogenetic relationships and serum neutralization was established among FCV isolates examined.

Amino Acid Sequence↗
PubMed2003

Endocytosis of adenovirus and adenovirus capsid proteins.

Key proteins of the icosahedral-shaped adenovirus (Ad) capsid mediate infection, and interact with cellular proteins to coordinate stepwise events of cell entry that produce successful gene transfer. Infection is mediated predominantly by the penton and fiber capsid proteins. The fiber initiates cell binding while the penton binds integrin coreceptors, triggering integrin-mediated endocytosis. Penton integrin signaling precedes viral escape from the endosomal vesicle. After cell binding, the virus undergoes stepwise disassembly of the capsid, shedding proteins during cell entry. Intracellular trafficking of the remaining capsid shell is mediated by the interaction of naked particles with the cytoskeleton. The capsid translocates toward the nucleus, with the majority of capsid proteins accumulating at the nuclear periphery, while viral DNA and associated protein VII are extruded through the nuclear pore. This discussion will encompass the current knowledge on Ad cell entry and trafficking, with an emphasis on the contribution of Ad capsid proteins to these processes. A greater understanding of the highly effective Ad cell entry pathway may lend itself to the development of safer drug and gene delivery alternatives utilizing similar pathways.

Adenoviridae↗
PubMed2005

Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography.

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.

Capsid↗
PubMed2006

Native hepatitis B virions and capsids visualized by electron cryomicroscopy.

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.

Animals↗
PubMed1984

Structure of the capsid of Kilham rat virus from small-angle neutron scattering.

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 X 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H2O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, we propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H2O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H2O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H2O. We propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

Amino Acids↗
PubMed2004

Evidence that the fully assembled capsid of Leishmania RNA virus 1-4 possesses catalytically active endoribonuclease activity.

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonuclease activity was found to be at 37(O)C to 42(O)C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.

Amino Acid Substitution↗
PubMed2000

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus.

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem-loop structure located 15-43 nt upstream of the 5' end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

Capsid↗
PubMed2006

Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor.

The host restriction factor TRIM5alpha mediates species-specific, early blocks to retrovirus infection; susceptibility to these blocks is determined by viral capsid sequences. Here we demonstrate that TRIM5alpha variants from Old World monkeys specifically associate with the HIV type 1 (HIV-1) capsid and that this interaction depends on the TRIM5alpha B30.2 domain. Human and New World monkey TRIM5alpha proteins associated less efficiently with the HIV-1 capsid, accounting for the lack of restriction in cells of these species. After infection, the expression of a restricting TRIM5alpha in the target cells correlated with a decrease in the amount of particulate capsid in the cytosol. In some cases, this loss of particulate capsid was accompanied by a detectable increase in soluble capsid protein. Inhibiting the proteasome did not abrogate restriction. Thus, TRIM5alpha restricts retroviral infection by specifically recognizing the capsid and promoting its rapid, premature disassembly.

Animals↗
PubMed2003

Nuclear import of hepatitis B virus capsids and release of the viral genome.

While studying the import of the hepatitis B virus genome into the nucleus of permeabilized tissue culture cells, we found that viral capsids were imported in intact form through the nuclear pore into the nuclear basket. Import depended on phosphorylation of the capsid protein and was mediated by the cellular transport receptors importin alpha and beta. Virus-derived capsids that contained the mature viral genome were able to release the viral DNA and capsid protein into the nucleoplasm. The uncoating reaction was independent of Ran, a GTP-binding enzyme responsible for dissociating other imported cargo from the inner face of the nuclear pore. Immature capsids that did not contain the mature viral genome reached the basket but did not release capsid proteins nor immature genomes into the nucleoplasm. The different fate of mature and immature capsids after passing the nuclear pore indicates that the outcome of a nuclear import event may be regulated within the nuclear basket.

Active Transport, Cell Nucleus↗
PubMed1990

Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.

Determination of icosahedral virion capsid size can be directly studied during helper-dependent lytic development of satellite P4 because the assembly pathway specified by the P2 helper virus is altered to yield smaller-sized capsids. Size determination (sid) mutations identify a P4-encoded function regulating this process. To determine whether the sid gene product is necessary and sufficient to redirect the assembly pathway, we (i) cloned the sid structural gene in a plasmid vector (pMA30) under the control of an inducible promoter and (ii) constructed a packaging substrate (pMA1), a P4 genome-sized plasmid containing only that region of P4, the cos site, necessary for encapsidation. Superinfection by P2 of a host carrying pMA30 under induced conditions resulted in a shift from large to small capsid production. P2 superinfection of a host carrying the cos plasmid pMA1 plus pMA30 under induced conditions yielded pMA1-transducing particles of P4 capsid size. These cloning-based analyses directly demonstrate that sid protein is the only P4 gene product required for small-capsid size determination. In the absence of the P2 O gene product no capsids of any size are assembled during solo infection by P2. Nevertheless, P2 Oam mutant superinfection of a host carrying pMA1 and pMA30 under induced conditions yielded small P4-sized transducing particles. We therefore propose that (i) the sid gene product competes with the O gene product to determine the assembly of small vs. large capsid sizes and (ii) both gene products probably function as temporary scaffolding proteins.

Base Sequence↗
PubMed2003

Central role of a serine phosphorylation site within duck hepatitis B virus core protein for capsid trafficking and genome release.

Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acid-supported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wild-type and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.

Alanine↗
PubMed1994

A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.

Amino Acid Sequence↗
PubMed1998

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.

Amino Acid Sequence↗
PubMed1995

Association of serum immunoglobulin G antibodies against human papillomavirus type 16 capsids with anal epidermoid carcinoma.

BACKGROUND: Anal epidermoid carcinoma is a relatively rare tumor, but its incidence has been increasing rapidly during the past few years. Genetic material from the major oncogenic types of human papillomavirus (HPV), types 16 and 18, has regularly been demonstrated in a substantial proportion of anal cancers, suggesting an etiologic role of HPV infection. Recently, serum antibodies against HPV type 16 capsids were shown to be a serologic measure of HPV16 infection. PURPOSE: We investigated whether serum antibodies against HPV16 capsids are associated with an increased risk of developing anal cancer. METHODS: Serum samples from 64 patients (48 women and 16 men) with untreated anal epidermoid cancer and from 79 age- and sex-matched healthy blood donors were analyzed for the levels of serum immunoglobulin G (IgG) against capsids of HPV16 by the enzyme-linked immunosorbent assay. The levels of serum IgG against HPV type 6 and bovine papillomavirus (BPV) capsids, as well as against HPV16 peptide antigens, were also measured. RESULTS: Whereas antibodies against HPV6 or BPV capsids were not significantly associated with anal cancer, the presence of IgG against HPV16 capsids exceeding the anti-BPV antibody levels was demonstrated among 55% (35 of 64) of the case patients but only among 4% (three of 79) of the control subjects (odds ratio [OR] = 30.4; 95% confidence interval [CI] = 8.4-161.5). Antibodies against HPV16 E2 and E7 peptides were also more common among case patients (OR = 12.8 and 95% CI = 5.4-31.5 for E2; OR = 3.0 and 95% CI = 1.4-6.7 for E7). CONCLUSION: The results suggest that HPV16 capsid antibodies are serologic markers for anal cancer. IMPLICATION: Exposure to HPV16 or related viruses appears to be a major risk factor in the majority of anal cancers.

Aged↗
PubMed1994

Mutations in the carboxy terminus of adeno-associated virus 2 capsid proteins affect viral infectivity: lack of an RGD integrin-binding motif.

Using site-directed mutagenesis, we tested whether a potential integrin-binding site, (composed of the amino acids RGD) which is predicted in the adeno-associated virus 2 (AAV-2) capsid open reading frame (ORF), plays a role in the infectivity of AAV-2. Nucleotide sequencing of wild-type and mutant capsid protein-coding sequences, however, revealed discrepancies with the published sequence data at several positions, including a frameshift in the carboxy terminus which cancels the RGD motif and extends the capsid ORF by 27 amino acids. This sequence was confirmed by protein sequencing of proteolytic fragments of VP3. Thus, the virus mutant (pTAV-p), in which the intention was to exchange D of the putative RGD motif for E, resulted in replacing I480 by S in the newly established ORF. A second virus mutant (pTAV-d), in which the intention was to delete the RGD peptide, in fact gave a shift into the ORF of the originally published sequence. The pTAV-p mutant showed a strongly reduced infectivity compared to wild-type AAV-2, whereas pTAV-d was not infectious at all. Neither mutant accumulated viral ssDNA as detected by Hirt extraction. Analysis of virus particle formation and subcellular localization of the capsid proteins revealed a defect of the mutant capsid proteins in capsid assembly. This shows that the newly established C-terminal sequence of the AAV capsid proteins plays an important role in viral assembly.

Amino Acid Sequence↗
PubMed1997

Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells.

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCI density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.

Amino Acid Sequence↗
PubMed2002

Studies on intracellular processing of the capsid protein of human astrovirus serotype 1 in infected cells.

Astroviruses are non-enveloped, positive-strand RNA viruses. Their structural (capsid) protein is processed extracellularly into several smaller fragments which are found on the mature viral particle. In addition, intracellular cleavage of the capsid protein has been proposed. However, analysis of capsid protein processing has been hampered by the lack of antibodies to regions near the N and C termini of the protein. Here we describe the construction of two infectious mutants of human astrovirus serotype 1 (HAstV-1), in which amino acids (aa) 11-30 or aa 783-787, respectively, of the 787 aa capsid protein were replaced by tag sequences. Processing of the tagged capsid proteins in infected Caco-2 cells was analysed by immunoprecipitation with specific reagents directed against the tags or against native internal regions of the capsid protein. No intracellular processing of the capsid protein in infected cells could be detected, while assembled viral particles were readily observed within cells.

Amino Acid Sequence↗