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PubMed1993

Virus protein assembly in microgravity.

The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid. VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure. Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly. Flight studies were conducted on STS-37 on April 5-9, 1991. Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0). After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation. Flight and ground-based samples were analyzed by electron microscopy. Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits. Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.

Animals↗
PubMed1979

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.

Capsid↗
PubMed1996

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Base Sequence↗
PubMed1974

Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D.

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.

Cell Line↗
PubMed1992

Normal modes of symmetric protein assemblies. Application to the tobacco mosaic virus protein disk.

We use group theoretical methods to study the molecular dynamics of symmetric protein multimers in the harmonic or quasiharmonic approximation. The method explicitly includes the long-range correlations between protein subunits. It can thus address collective dynamic effects, such as cooperativity between subunits. The n lowest-frequency normal modes of each individual subunit are combined into symmetry coordinates for the entire multimer. The Hessian of the potential energy is thereby reduced to a series of blocks of order n or 2n. In the quasiharmonic approximation, the covariance matrix of the atomic oscillations is reduced to the same block structure by an analogous set of symmetry coordinates. The method is applied to one layer of the tobacco mosaic virus protein disk in vacuo, to gain insight into the role of conformational fluctuations and electrostatics in tobacco mosaic virus assembly. The system has 78,000 classical, positional, degrees of freedom, yet the calculation is reduced by symmetry to a problem of order 4,600. Normal modes in the 0-100 cm-1 range were calculated. The calculated correlations extend mainly from each subunit to its nearest neighbors. The network of core helices has weak correlations with the rest of the structure. Similarly, the inner loops 90-108 are uncorrelated with the rest of the structure. Thus, the model predicts that the dielectric response in the RNA-binding region is mainly due to the loops alone.

Binding Sites↗
PubMed1985

Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex.

The effect of the carboxylic ionophore monensin on the maturation of Uukuniemi virus, a bunyavirus, and the transport of its two membrane glycoproteins, G1 and G2, were studied in chicken embryo fibroblasts and baby hamster kidney cells. Virus maturation, which occurs in the Golgi complex (E. Kuismanen, K. Hedman, J. Saraste, and R. F. Pettersson, Mol. Cell. Biol. 2:1444-1458, 1982; E. Kuismanen, B. Bång, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984), was effectively inhibited by the drug (1 or 10 microM) as studied by electron microscopy and by assaying the release of infectious or radiolabeled virus. Immunoelectron microscopy showed that association of viral nucleocapsids with the cytoplasmic surface of glycoprotein-containing Golgi membranes, a prerequisite for virus budding, was unaffected by monensin. In the presence of the drug, the virus glycoproteins assembled into long, tubular structures extending into the lumen of Golgi-derived vacuoles, suggesting that monensin inhibited a terminal step in the assembly of the virus. Intracellular transport and expression of the virus membrane glycoproteins G1 and G2 at the cell surface were not inhibited by monensin as studied by immunocytochemical and radiolabeling techniques. Pulse-chase experiments in the presence of monensin showed that intracellular G1 acquired only partially endo-H-resistant glycans. The sialylation of G1 appearing on the cell surface in the presence of the drug was decreased, whereas sialylation of G2 apparently was inhibited to a lesser extent, as shown by external labeling of the cells with the periodate-boro[3H]hydride method. Thus, monensin exerted a differential effect on the terminal glycosylation of G1 and G2. Unlike several membrane and secretory glycoproteins, both G1 and G2 could enter a functional transport pathway in the presence of monensin and become expressed at the cell surface.

Animals↗
PubMed2001

Functional reconstitution of lost activity of chemically cross-linked or mutant moloney murine leukemia virus nucleocapsid proteins by trans-complementation.

Intermolecular crosslinking of the retroviral gag structural proteins, capsid (CA) and nucleocapsid (NC), by oxidation of cysteine thiols interferes with virus assembly and infectivity. PD156202 is a dithiobisbenzamide with broad antiviral activity against retroviruses. Treatment of cell free Moloney murine leukemia virus (MuLV) particles with PD156202 induced crosslinking of gag structural proteins and inhibited reverse transcription in mellitin permeabilized virions. Site directed mutagenesis of NC cysteines in the zinc finger of MuLV proviral DNA resulted in virus particles that were noninfectious. The NC mutant virus did not display any intermolecular crosslinking following treatment with PD156202 under nonreducing conditions, which supported that NC cysteine residues participated in PD156202 mediated crosslinking. Replication of MuLV NC mutant virus could be restored by independent expression of wild type NC, making it susceptible to PD156202. These results demonstrate that oxidation of NC cysteines are detrimental for virus assembly and that complementation with wild type NC restored the nucleocapsid protein activity.

3T3 Cells↗
PubMed2003

Snow Mountain virus genome sequence and virus-like particle assembly.

Snow Mountain virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. In this study, the positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3' polyadenylated tract. The genome is organized into three open reading frames typical of caliciviruses in the Norovirus genus. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other genogroup II noroviruses, and most closely related to GII strains Melksham and Hawaii virus. In addition, comparative sequence analyses indicated that SMV is likely a recombinant norovirus. VP1/VP2 proteins self-assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1, but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly under standard conditions of expression.

Caliciviridae↗
PubMed1996

High resolution analysis of the readthrough domain of beet necrotic yellow vein virus readthrough protein: a KTER motif is important for efficient transmission of the virus by Polymyxa betae.

The 5'-terminal cistron of beet necrotic yellow vein furovirus RNA 2 encodes the 21 kDa major viral coat protein and terminates with an amber stop codon which can undergo suppression to give rise to a 75 kDa readthrough (RT) protein referred to as P75. P75 is a minor component of virions and the 54 kDa RT domain following the coat protein sequence is important both for virus assembly and transmission by the fungal vector Polymyxa betae. To better define the regions of the RT domain involved in these two steps, RNA 2 transcripts encoding different in-frame RT domain deletion mutants were tested for their ability to form virions when inoculated to plants with the other viral RNAs and to be fungus-transmitted. All deletions in the N-terminal half of the RT domain interfered with virus assembly and partially or completely inhibited fungus transmission. A 4 1 1 nucleotide deletion within the C-terminal half of the RT domain did not inhibit assembly but blocked fungus transmission of the virus. Alanine scanning mutagenesis within the aforesaid 4 1 1 nucleotide subdomain identified a peptide motif (KTER) which is important for the fungus transmission process.

Base Sequence↗
PubMed2001

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

3T3 Cells↗
PubMed1999

Mutational analysis, using a full-length rubella virus cDNA clone, of rubella virus E1 transmembrane and cytoplasmic domains required for virus release.

We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.

Animals↗
PubMed2004

A tiling approach to virus capsid assembly explaining a structural puzzle in virology.

A novel approach for the description of the protein stoichiometry of viral capsids, that is the protein shells protecting the viral genome, is introduced based on tiling theory. This approach generalizes Caspar-Klug theory of quasi-equivalence to account also for non-quasi-equivalent subunit arrangements in icosahedral virus capsids that have been observed experimentally but are not covered by the Caspar-Klug approach. In particular, the new approach is used to explain the structure of polyoma virus, Simian Virus 40 and L-A virus capsids, which are considered structural puzzles in view of Caspar-Klug theory.

Capsid↗
PubMed2003

Functional replacement of nucleocapsid flanking regions by heterologous counterparts with divergent primary sequences: effects of chimeric nucleocapsid on the retroviral replication cycle.

Nucleocapsid (NC) proteins in most retroviruses have a well-conserved Cys-His box(es) as well as more divergent flanking regions that are rich in basic residues. Mutations in the flanking regions can affect RNA packaging, virus assembly, and reverse transcription of the viral RNA. To gain a further understanding of the roles of NC flanking regions in the retroviral replication cycle, we generated and characterized chimeric gag-pol expression constructs derived from murine leukemia virus and spleen necrosis virus by replacing an NC flanking region from one virus with the counterpart from the other virus. We found that all four chimeras were able to generate virions, package viral RNA, and complete the viral replication cycle. Two chimeras had mild defects in virus assembly that correlated with a decrease in the isoelectric points of NCs, suggesting that the basic nature of NC is important in virus assembly. This finding indicates that, although the primary sequences of these flanking regions have little homology, the heterologous sequences are functional both as part of the Gag polyprotein and as processed NC protein.

Amino Acid Sequence↗
PubMed2004

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

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

Algorithms↗
PubMed2003

Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins.

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75 degrees C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.

Capsid↗
PubMed1998

Nucleotide sequence of the 5'-terminus of Newcastle disease virus and assembly of the complete genomic sequence: agreement with the "rule of six".

We have determined the sequences of the 5' ends of three strains of Newcastle disease virus, permitting the assembly of the entire genomic sequence, which amounts to 15,186 nucleotides. This length is in agreement with the rule of six, which has been shown to determine replication efficiency in similar viruses. Comparison of the extreme 5' end of the trailer sequence with that of the 3'-terminal leader sequence of the virus reveals a high degree of complementarity. Variation between the 5'-terminal sequences of the different strains reveals the presence of alternative L gene polyadenylation signals, leading to correspondingly different trailer lengths.

Animals↗
PubMed1982

In vivo and in vitro models of demyelinating diseases. V. Comparison of the assembly of mouse hepatitis virus, strain JHM, in two murine cell lines.

The developmental sequence of a neurotropic strain (JHM) of mouse hepatitis virus was examined by transmission electron microscopy and immunocytology. The nucleoprotein core of this coronavirus, which contains RNA of positive polarity and is helical in configuration, becomes incorporated into enveloped particles in the same manner as the nucleocapsids of the orthomyxo- and paramyxoviruses. However, JHM virus is assembled intracellularly by budding at surfaces of smooth membranous vacuoles. A comparison of JHM virus replication in L2 and 17Cl-1 cell lines revealed that L2 cells undergo more rapid cytopathology and cease virus production much sooner than 17Cl-l cells. In L2 cells the accumulation of core material appears to continue after the abrupt cessation of virus assembly. This is evident by the massive cytoplasmic accumulation of structure resembling nucleocapsids, which react with hybridoma antibody to the nucleocapsid antigen as demonstrated by the immunoperoxidase procedure. The current findings are consistent with our previously published demonstration, using cells of neural and other deviation, of the fundamental role of the host cell type in regulating the replication and expression of coronaviruses.

Animals↗