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At least 19 recordsLinked to original sources
PubMed1992

Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1).

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.

Amino Acid Sequence↗
PubMed1976

Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.

Amino Acid Sequence↗
PubMed1979

Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.

Kinetics↗
PubMed1992

In vitro mutagenesis of biologically active transcripts of beet necrotic yellow vein virus RNA 2: evidence that a domain of the 75-kDa readthrough protein is important for efficient virus assembly.

RNA 2 of the multipartite genome of beet necrotic yellow vein virus carries the cistron for 21-kDa viral coat protein at its 5' extremity. The amber termination codon of the coat protein cistron undergoes suppression approximately 10% of the time so that translation continues into an adjacent 54-kDa open reading frame, yielding a 75-kDa readthrough protein. The roles of coat protein and the readthrough protein in infection were investigated with biologically active transcripts of RNA 2. Much of the coat protein cistron of the RNA 2 transcript could be deleted without interfering with viral replication and local lesion formation on leaves, although formation of the rod-shaped virions did not occur. Mutants in which the amber coat protein termination codon was replaced with an ochre codon or a tyrosine codon were also viable. The ochre codon was suppressed both in vitro and in planta. The mutant containing the tyrosine substitution produced only the 75-kDa read-through protein and was deficient in viral assembly. Deletions in the 54-kDa readthrough domain were also viable in planta but had different effects on virus assembly. A deletion in the C-terminal portion of the readthrough domain did not interfere with RNA packaging but, unexpectedly, deletions in the N-terminal portion were assembly deficient, although 21-kDa coat protein was produced in planta. Thus, the 75-kDa protein can apparently intervene in virion assembly even though it has not been detected in purified virions.

Base Sequence↗
PubMed1992

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.

Amino Acid Sequence↗
PubMedDate not supplied

[Model for tobacco mosaic virus assembly in vitro: specific dislocation by cooperative single-letter purine recognition and non-cooperative RNA "locking" between subunit layers].

A model of the molecular mechanism of tobacco mosaic virus (TMV) assembly in vitro is proposed. The model is based on the assumption that on interaction of TMV RNA chain with a double protein disk two different types of structural alterations occur in the protein subunits: "dislocating alterations" leading to screw dislocation of the disk into a helix fragment and "locking alterations" leading to locking of the RNA chain between the subunits layers. During initiation of TMV assembly the dislocating alterations occur cooperatively in all subunits of the disk as a result of specific interaction of purines (guanines) in the middle (horizontal) position of every trinucleotide unit with the 32--39 loop of the TMV protein molecule. Locking alterations are induced non-cooperatively by phosphate groups and lateral (vertical) bases of trinucleotide units. The sequence of events during TMV assembly in vitro and the role of topological factors in this process are discussed.

Models, Biological↗
PubMed1992

The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly.

Assembly of replication-competent hepatitis B virus (HBV) nucleocapsids requires the interaction of the core protein, the P protein, and the RNA pregenome. The core protein contains an arginine-rich C-terminal domain which is dispensable for particle formation in heterologous expression systems. Using transient expression in HuH7 cells of a series of C-terminally truncated core proteins, I examined the functional role of this basic region in the context of a complete HBV genome. All variants containing at least the 144 N-terminal amino acids were assembly competent, but efficient pregenome encapsidation was observed only with variants consisting of 164 or more amino acids. These data indicate that one function of the arginine-rich region is to provide the interactions between core protein and RNA pregenome. However, in cores from the variant ending with amino acid 164, the production of complete positive-strand DNA was drastically reduced. Moreover, almost all positive-strand DNA originated from in situ priming, whereas in wild-type particles, this type of priming not supporting the formation of relaxed circular DNA (RC-DNA) accounted for about one half of the positive strands. Further C-terminal residues to position 173 restored RC-DNA formation, and the corresponding variant did not differ from the full-length core protein in all assays used. The observation that RNA encapsidation and formation of RC-DNA can be genetically separated suggests that the core protein, via its basic C-terminal region, also acts as an essential auxiliary component in HBV replication, possibly like a histone, or like a single-stranded-DNA-binding protein. In contrast to their importance for HBV replication, sequences beyond amino acid 164 were not required for the formation of enveloped virions. Since particles from variant 164 did not contain mature DNA genomes, a genome maturation signal is apparently not required for HBV nucleocapsid envelopment.

Amino Acid Sequence↗
PubMed1992

Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame.

The herpes simplex virus (HSV) type 1 assembly protein ICP35 consists of a family of polypeptides, ranging in molecular weight from about 45,000-39,000. The lower molecular weight forms of ICP35 are derived from the higher molecular weight species by slow post-translational modification. The reading frame of gene UL26 and the region within this gene which exhibited homology to the cytomegalovirus assembly protein, the analogous protein to ICP35, were expressed separately under immediate-early (IE) gene regulation in a HSV vector containing a temperature-sensitive mutation in the major transcriptional regulator Vmw175. Monoclonal antibody specific for ICP35 immunoprecipitated several polypeptides with molecular weights around 75,000 from extracts of cells infected with a recombinant expressing the IE gene UL26 at the nonpermissive temperature (NPT). These results suggested that the UL26 gene specified a protein distinct from ICP35 but which had some antigenic sites in common with ICP35. In extracts of cells infected at the NPT with a recombinant expressing only the carboxy terminal half of UL26 coding sequences, the monoclonal antibody immunoprecipitated large amounts of the high molecular weight forms of ICP35. The lower molecular weight processed forms of ICP35, however, were not detectable. When cells were coinfected with both recombinants ICP35 was processed to its lower molecular weight forms. This processing step, which occurred near the carboxy terminus of ICP35, was not dependent on capsid formation. The work, together with previous information on the processing of the CMV assembly protein, suggests that UL26 product may be a protease.

Animals↗
PubMed1975

Virus assembly.

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Adenoviridae↗
PubMed1991

Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding.

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and E1 appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.

Amino Acid Sequence↗