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PubMed1999

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins.

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

Amino Acid Sequence↗
PubMed1997

Assemblases and coupling proteins in thick filament assembly.

Thick filaments are stable assemblies of myosin that are characteristic of specific muscle types from both vertebrates and invertebrates. In general, their structure and assembly require remarkably precise determination of lengths and diameters, structural differentiation and nonequivalence of myosins, a high degree of inelasticity and rigidity, and dynamic regulation of assembly and disassembly in response to both extracellular and intracellular signals. Directed assembly of myosin in which additional proteins function in key roles, therefore, is more likely to be significant than the simple self assembly of myosin into thick filaments. The nematode Caenorhabditis elegans permits a wide spectrum of biochemical, genetic, molecular and structural approaches to be applied to the experimental testing of this hypothesis. Biochemical analysis of C. elegans thick filaments reveals that paramyosin, a homologue of the myosin rod that is the unique product of a single genetic locus, exists as two populations which differ by post-translational modification. The major paramyosin species interacts with the two genetically specified myosin heavy chain isoforms. The minor paramyosin species is organized within the cores of the thick filaments, where it is associated stoichiometrically with three recently identified proteins P20, P28 and P30. These proteins have now been characterized molecularly and contain unique, novel amino acid sequences. Structural analysis of the core shows that seven paramyosin subfilaments are crosslinked by additional internal proteins into a highly rigid tubule. P20, P28 and P30 are proposed to couple the paramyosin subfilaments together into the core tubule during filament assembly. Mutants that affect paramyosin assembly are being characterized for alterations in the core proteins. A fourth protein has been identified recently as the product of the unc-45 gene. Computational analysis of this gene's DNA suggests that the predicted protein may exhibit protein phosphatase and chaperone activities. Genetic analysis shows that three classes of specific unc-45 mutant proteins differentially interact with the two myosins during thick filament assembly. The unc-45 protein is proposed to be a myosin assemblase, a protein catalyst of thick filament assembly.

Actin Cytoskeleton↗
PubMed2005

Simulation study of the contribution of oligomer/oligomer binding to capsid assembly kinetics.

The process by which hundreds of identical capsid proteins self-assemble into icosahedral structures is complex and poorly understood. Establishing constraints on the assembly pathways is crucial to building reliable theoretical models. For example, it is currently an open question to what degree overall assembly kinetics are dominated by one or a few most efficient pathways versus the enormous number theoretically possible. The importance of this question, however, is often overlooked due to the difficulties of addressing it in either theoretical or experimental practice. We apply a computer model based on a discrete-event simulation method to evaluate the contributions of nondominant pathways to overall assembly kinetics. This is accomplished by comparing two possible assembly models: one allowing growth to proceed only by the accretion of individual assembly subunits and the other allowing the binding of sterically compatible assembly intermediates any sizes. Simulations show that the two models perform almost identically under low binding rate conditions, where growth is strongly nucleation-limited, but sharply diverge under conditions of higher association rates or coat protein concentrations. The results suggest the importance of identifying the actual binding pattern if one is to build reliable models of capsid assembly or other complex self-assembly processes.

Biophysics↗
PubMed1988

Characteristics of work actions of shoe manufacturing assembly line workers and a cross-sectional factor-control study on occupational cervicobrachial disorders.

The characteristic work actions of female shoe manufacturing assembly line workers were analyzed by the records of 8-mm cine-films. The relationship between cervicobrachial disorders and work actions was investigated as a cross-sectional factor control study by using sex-age matched pairs for non-assembly line workers (102 pairs). The following conclusions were obtained: 1) The assembly line workers handled about 3,400 sneaker shoes per day on the assembly line. A completed shoe weighed 200-500 g. The metal last weighted 400-1,200 g. As the lines were not completely mechanized, the workers passed shoes to the next worker by hand. 2) In the line selected for the study of work actions, 28 female workers and one male worker were engaged. The work direction of the line was one-way (from left to right or vice versa). The actions of the workers were classified into four fundamental actions: i) grasping the shoe or tool, ii) extending or iii) bending of the arms, and iv) keeping the arms in a certain position. These fundamental actions were repeated more than 3,400 times per day by each worker. The time spent in holding a shoe in the left hand was longer than that of the right hand in holding a shoe or tool. 3) Results of medical examinations showed a higher prevalence in assembly line workers than that in non-assembly line workers. Especially the prevalence rate of tapping test, pain sensibility test, vibratory sensibility test, Morley's test, tenosynovitis in the fingers, tenderness at spinal muscle around the thoracic vertebrae, levator muscle of scapula, trapezius muscle, rhomboid muscle, infraspinatus muscle, greater pectoral muscle, anterior scalene muscle, thenar eminence, biceps muscle of arm, brachioradial muscle, and antebrachial flexor muscle were found to be different significantly by McNemar's test between the two groups. These disorders were appeared in the left shoulder, arm and hand. 4) As the non-assembly line workers were not engaged in compulsory work or in one-way work direction, they injured the right side (skillful side). On the contrary, it is considered that compulsory transfer of shoes and one-way work direction imposes a heavier load on the left side of the body in assembly line workers, and consequently they injured the left side more severely. 5) It is concluded that the sustained task of handing over shoes to the next worker or one-way work direction caused cervicobrachial disorders of assembly line workers, especially on the left side of the body.

Adult↗
PubMed1997

Impact of a truncated invariant chain on in vitro assembly of class II MHC molecules depends on the affinity of invariant chain for a given alpha beta dimer.

The assembly of major histocompatibility complex (MHC) class II alpha and beta chains occurs in the endoplasmic reticulum (ER) with the involvement of MHC class II-associated invariant chain (Ii). The present study investigated the impact of Ii on the assembly of both I-A haplotype-matched and -mismatched alpha and beta chains using an in vitro translation system. The alpha and beta chains of I-Ab, I-Ad and I-Ak were cotranslated in vitro in different combinations with or without cotranslation of a truncated murine Ii (mIi 1-131). The translated products were sequentially immunoprecipitated, first with conformation-dependent monoclonal antibodies, then with conformation-independent antibodies. The results show: (1), Ii did not associate with free A alpha and free A beta chains; (2), mIi 1-131 significantly augmented the amount of properly assembled A alpha b A beta b, A alpha b A beta d, A alpha b A beta k and A alpha k A beta b dimers, but had little affect on the assembly of A alpha d A beta d, A alpha k A beta k, A alpha d A beta b, A alpha k A beta d and A alpha d A beta k; (3), All A alpha A beta dimers whose assembly could be significantly facilitated by mIi 1-131 could be coimmunoprecipitated along with substantial amounts of mIi 1-131. This finding is consistent with prior observations that the impact of Ii on class II molecule assembly is allele specific. Furthermore, these results suggest that the efficient assembly of alpha and beta chains is primarily determined by the affinity between alpha and beta chains and the the high affinity of mIi for A alpha A beta dimers is required for mIi 1-131 to assist proper A alpha A beta assembly, most probably through a mechanism in which Ii stabilizes properly assembled A alpha A beta dimers or promotes folding of associated alpha and beta chains to help achieve a stable dimer state.

Animals↗
PubMed1991

Gas and steam sterilization of assembled versus disassembled laparoscopic equipment. Microbiologic studies.

Current recommendations specify disassembly of most laparoscopic equipment prior to sterilization. Surgical technicians, however, are often unfamiliar with the proper assembly of laparoscopic instruments, resulting in possible patient injury from equipment malfunction. Therefore, we tested the hypothesis that disassembled laparoscopic equipment is sterilized more thoroughly than assembled equipment. We inoculated internal sites on laparoscopic instruments prior to assembly with bacterial spores resistant to ethylene oxide and steam sterilization. We also manually cleaned the equipment after inoculation prior to steam sterilization of both the assembled and disassembled instruments. The control instruments were stored at room temperature during test sterilization runs. No vegetative bacteria survived ethylene oxide or steam sterilization in assembled equipment, but despite a significant reduction, spore-forming bacteria could be cultured from the assembled equipment. If the instruments were washed before steam sterilization, there was similar spore clearance in the assembled and disassembled instruments, with both groups attaining a high level of disinfection. Our data suggest that disassembly, cleaning and proper assembly of equipment prior to sterilization present no more risk of infection transmission than does disassembly during prevacuum steam sterilization. The method provides properly assembled and functioning equipment at the time of surgery.

Clinical Protocols↗
PubMed1989

Increased assembly of clathrin occurs in response to mitogenic activation of murine lymphocytes.

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.

Animals↗
PubMed1987

Formation of the alpha-bungarotoxin binding site and assembly of the nicotinic acetylcholine receptor subunits occur in the endoplasmic reticulum.

During the process by which newly synthesized subunits of the nicotinic acetylcholine receptor (stoichiometry = alpha 2 beta gamma delta) mature and acquire the properties of the fully functional cell surface receptor, they undergo numerous covalent and noncovalent modifications. Using ligand-mediated and subunit-specific immunoprecipitation, four forms in the maturation of the alpha subunit can be detected: the primary translation product; alpha subunit that can bind alpha-bungarotoxin; alpha subunit assembled with the other subunits; and surface receptor. The alpha subunit acquires the ability to bind alpha-bungarotoxin with a t1/2 of approximately 40 min after translation and becomes assembled with a t1/2 of 80 min after translation. Using metabolic labeling and sucrose gradient fractionation, we have determined the subcellular location of alpha subunit when it acquires the ability to bind alpha-bungarotoxin and when it is assembled. Golgi membranes were identified across the gradient by the enzymatic activities UDP-galactose:N-acetylglucosamine galactosyltransferase and alpha-mannosidase. Endoplasmic reticulum membranes were identified by the enzymatic activity glucose-6-phosphatase and by the presence of newly synthesized alpha and beta subunits. Pulse-labeled alpha subunit that bound alpha-bungarotoxin was first detected co-migrating in the gradient with the glucose-6-phosphatase activity. Therefore, the capacity to bind alpha-bungarotoxin was acquired while the alpha subunit was in the endoplasmic reticulum. Assembled alpha subunit was detected by immunoprecipitating with an anti-beta subunit-specific monoclonal antibody. By this method, assembled receptor was first detected 15 min after translation in both the endoplasmic and Golgi portions of the gradient. To validate this method of detecting assembled receptor, we determined the sedimentation coefficient of the receptor subunits in the endoplasmic reticulum. Both unassembled subunits with sedimentation coefficients of 5 S and assembled receptor with a sedimentation coefficient of 9 S were recovered from the endoplasmic reticulum portion of the gradient. Thus, our data concerning the subcellular site of assembly are consistent with assembly occurring in the endoplasmic reticulum followed by rapid transport to the Golgi.

Binding Sites↗
PubMed1993

Partial assembly of the yeast vacuolar H(+)-ATPase in mutants lacking one subunit of the enzyme.

Partial assembly of the peripheral and integral membrane sectors of the yeast vacuolar H(+)-ATPase has been detected in mutants lacking one subunit of the enzyme. Assembled complexes of the vacuolar H(+)-ATPase could be immunoprecipitated from biosynthetically labeled wild-type cells using monoclonal antibodies specific for the 69- and 60-kDa subunits of the enzyme, and assembled membrane (V0) sectors could be immunoprecipitated using a monoclonal antibody against the 100-kDa subunits. Parallel immunoprecipitations from mutant cells lacking one subunit of the vacuolar H(+)-ATPase revealed different degrees of assembly depending on the subunit that was missing. Partially assembled complexes of the peripheral subunits could also be detected in a soluble, cytoplasmic fraction from wild-type and mutant cells following glycerol gradient fractionation. The results indicate that the peripheral (V1) sector and integral membrane (V0) sectors of the yeast vacuolar H(+)-ATPase can assemble independently. The 69-, 60-, and 27-kDa subunits all appear to be necessary for any assembly of the V1 sector to occur, but these subunits and the 32-kDa subunit can assemble into a complex in the absence of the 42-kDa peripheral subunit. The implications of the results for the structure and assembly of the yeast vacuolar H(+)-ATPase are discussed.

Centrifugation↗
PubMed2002

Dynamics of ATP-dependent chromatin assembly by ACF.

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases. Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2#150;4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA. These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.

Adenosine Triphosphate↗
PubMed2001

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Animals↗
PubMed1998

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo.

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.

Bacterial Proteins↗
PubMed2001

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.

Bacteriophage P22↗
PubMed2006

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles.

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.

Animals↗
PubMedDate not supplied

Modeling virus self-assembly pathways: avoiding dynamics using geometric constraint decomposition.

We develop a model for elucidating the assembly pathways by which an icosahedral viral shell forms from 60 identical constituent protein monomers. This poorly understood process a remarkable example of macromolecular self-assembly occuring in nature and possesses many features that are desirable while engineering self-assembly at the nanoscale. The model uses static geometric and tensegrity constraints to represent the driving (weak) forces that cause a viral shell to assemble and hold it together. The goal is to answer focused questions about the structural properties of a successful assembly pathway. Pathways and their properties are carefully defined and computed using computational algebra and geometry, specifically state-of-art concepts in geometric constraint decomposition. The model is analyzable and refinable and avoids expensive dynamics. We show that it has a provably tractable and accurate computational simulation and that its predictions are roughly consistent with known information about viral shell assembly. Justifications for mathematical and biochemical assumptions are provided, and comparisons are drawn with other virus assembly models. A method for more conclusive experimental validation involving specific viruses is sketched. Overall, the paper indicates a strong and direct, mutually beneficial interplay between (a) the concepts underlying macromolecular assembly; and (b) a wide variety of established as well as novel concepts from combinatorial and computational algebra, geometry and algebraic complexity.

Computer Simulation↗
PubMed2006

Alphavirus capsid protein helix I controls a checkpoint in nucleocapsid core assembly.

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.

Alphavirus↗
PubMed1998

Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain.

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr559gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.

Animals↗
PubMed2000

Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape.

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

Amino Acids↗