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PubMed1998

Type D retrovirus capsid assembly and release are active events requiring ATP.

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-D-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Adenosine Triphosphate↗
PubMed2004

Domain structures and roles in bacteriophage HK97 capsid assembly and maturation.

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.

Asparagine↗
PubMed2005

The requirements and mechanism for capsid assembly and budding of bovine foamy virus.

Little is known about assembly of non-primate foamy virus (FV) such as bovine foamy virus (BFV). To help determine the requirements for assembly of BFV, we constructed BFV-Gag expression plasmids containing all or part of the gag gene, with or without modification by addition of myristate (Myr). Each construct was transfected alone, and with pFenv, into Sf-9 insect cells. The results showed that only the entire Gag could transit through nucleus, which is required for BFV viral assembly in the cytoplasm. Unlike other retroviruses (but like primate foamy viruses), BFV requires the coexpression of the Env protein for viral particle budding. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to lack of a functional ER retrieval signal (ERRS). The results also showed that addition of a Myr-membrane targeting signal to the C-terminus of Gag could restore the budding from plasma membrane, implying that Myr-membrane targeting signal could substitute for Env protein in budding.

Animals↗
PubMed2003

HIV capsid assembly.

HIV Gag assembly is the first and most essential step in the formation of virus particles. Following protein synthesis, Gag relocates from ribosomes and forms a virus particle at the plasma membrane, using host factors and machinery. Early studies focused on mapping the regions within Gag required for assembly and identified three distinct domains (M, I, and L), although their precise locations within the three-dimensional structure of Gag awaited later study. In this review, I summarize the mapping results in the light of recent progress on Gag structures made by nuclear magnetic resonance and X-ray crystallography as well as further functional analysis. These data are largely consistent and provide sufficient information for an understanding of the interactions and functions of the assembly domains at a macromolecular level. Current studies have moved on to the identification of the host factors and machinery used in the process of Gag assembly. Cumulative data suggest that the dynamics of Gag assembly and transport are achieved not by simply using, but rather by taking control of, cellular machinery. Key area in the process include interactions with TSG101, L domain receptor which normally functions in the endosomal sorting pathway and with lipid rafts, a type of M domain receptor, which has been suggested to be the sites for effective concentration of Gag. The review provides a summary of these data and discusses the likely direction of future studies.

Capsid Proteins↗
PubMed2003

Implication of the product of the bovine herpesvirus type 1 UL25 gene in capsid assembly.

The UL25 ORF of bovine herpesvirus type 1 (BHV-1) was demonstrated recently to represent a gene encoding a 63 kDa viral protein. To investigate the role of this gene in virus replication, a BHV-1 UL25 deletion mutant was constructed. Although the UL25 mutant synthesizes late viral proteins and viral DNA, it fails to produce virus progeny in cells that do not express the UL25 gene, demonstrating that the UL25 protein is essential for the replicative cycle of BHV-1. Moreover, Southern blotting analyses of HindIII-digested DNA from infected non-complementing cells probed with the leftward terminal fragment of the BHV-1 linear genome revealed that the cleavage of the viral DNA produced is not impaired. However, the packaging of this cleaved DNA is compromised severely, since only few full C capsids were observed in infected non-complementing cells by transmission electron microscopy.

Animals↗
PubMed1998

The molecular basis of HIV capsid assembly.

In common with many aspects of the HIV life cycle, the assembly of the virus particle has been the subject of intense investigation over recent years. Study of the subject is facilitated by the fact that only a single gene product, the Pr55 Gag protein, is required for virus assembly. A combination of site directed mutagenesis, biochemical characterisation and structural studies have led to a picture of the overall architecture of the particle, the partial structure of Pr55, and the subdomains involved in oligomerisation. Copyright 1998 John Wiley & Sons, Ltd.

Journal Article↗
PubMed2000

A cell-line-specific defect in the intracellular transport and release of assembled retroviral capsids.

Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled form (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the development of a recombinant baculovirus/insect cell system for the expression of both C-type and D-type Gag polyproteins, we discovered an insect cell line (High Five) with two distinct defects that resulted in the reduced release of virus-like particles. The first of these was a pronounced defect in the transport of D-type but not C-type Gag polyproteins to the plasma membrane. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) Gag precursors accumulate assembled immature capsids in large cytoplasmic aggregates similar to a transport-defective mutant (MA-A18V). In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogenesis mutant (MA-R55W) and those of human immunodeficiency virus were transported to the plasma membrane for assembly and budding of virions. When pulse-labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequestered in a higher-molecular-mass complex. Compared to Sf9 insect cells, the High Five cells also demonstrate a defect in the release of C-type virus particles. These findings support the hypothesis that host cell factors are important in the process of Gag transport and in the release of enveloped viral particles.

Animals↗
PubMed2005

Electrostatic interactions govern both nucleation and elongation during phage P22 procapsid assembly.

Icosahedral capsid assembly is an example of a reaction controlled solely by the interactions of the proteins involved. Bacteriophage P22 procapsids can be assembled in vitro by mixing coat and scaffolding proteins in a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here, we investigated the effect of the buffer composition on the interactions necessary for capsid assembly. Different concentrations of various salts, chosen to follow the electroselectivity series for anions, were added to the assembly reaction. The concentration and type of salt was found to be crucial for proper nucleation of procapsids. Nucleation in low salt concentrations readily occurred but led to bowl-like partial procapsids, as visualized by negative stain electron microscopy. The edge of the partial capsids remained assembly-competent since coat protein addition triggered procapsid completion. The addition of salt to the partial capsids also caused procapsid completion. In addition, each salt affected both assembly rates and the extent of procapsid formation. We hypothesize that low salt conditions increase the coat protein:scaffolding protein affinity, causing excessive nuclei to form, which decreases coat protein levels leading to incomplete assembly.

Bacteriophage P22↗
PubMed1982

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.

Capsid↗
PubMed2004

Conservation of a stepwise, energy-sensitive pathway involving HP68 for assembly of primate lentivirus capsids in cells.

Previously we have described a stepwise, energy-dependent pathway for human immunodeficiency virus type 1 (HIV-1) capsid assembly in a cell-free system. In this pathway, Gag polypeptides utilize the cellular factor HP68 and assemble into immature capsids by way of assembly intermediates that have defined biochemical characteristics. Here we address whether this pathway is universally conserved among primate lentiviruses and can be observed in mammalian cells. We demonstrate that HIV-2 Gag associates with human HP68 in a cell-free system and that Gag proteins of HIV-2, simian immunodeficiency virus SIVmac239, and SIVagm associate with endogenous HP68 in primate cells, as is seen for HIV-1. Analysis of primate cells expressing lentivirus Gag proteins revealed Gag-containing complexes with the same sedimentation values as seen for previously described HIV-1 assembly intermediates in the cell-free system (10S, 80-150S, and 500S). These complexes fit criteria for assembly intermediates as judged by energy sensitivity, pattern of HP68 association, and the failure of specific complexes to be formed by assembly-incompetent Gag mutants. We also demonstrate that virus-like particles released from cells do not appear to contain HP68, suggesting that HP68 is released from Gag upon completion of capsid assembly in cells, as was observed previously in the cell-free system. Together these findings support a model in which all primate lentivirus capsids assemble by a conserved pathway of HP68-containing, energy-dependent assembly intermediates that have specific biochemical features.

ATP-Binding Cassette Transporters↗
PubMed1990

Intragenic suppression of a capsid assembly-defective P22 tailspike mutation.

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsid↗
PubMed1994

Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses.

Immature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence of VP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.

Animals↗
PubMed2005

Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro.

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.

Capsid Proteins↗
PubMed1984

Characterisation of a herpes simplex virus type 1 mutant which has a temperature-sensitive defect in penetration of cells and assembly of capsids.

A herpes simplex virus type 1 (HSV-1) mutant, ts1204, which has a temperature-sensitive (ts) mutation located within genome map coordinates 0.318 to 0.324, close to but outside the coding sequences of the glycoprotein gB gene, has been characterised. Although this mutant adsorbed to the cell surface at the nonpermissive temperature (NPT), it failed to penetrate the cell membrane. As a consequence of this defect, high multiplicities of infection of ts1204 blocked subsequent infection of cells by wild-type HSV-1. By contrast, at the NPT, superinfection of cells with HSV-2 was not inhibited by prior infection with ts1204. The penetration defect could be overcome either by brief incubation of mutant virus-infected cells at the permissive temperature, or by treatment of the cells with polyethylene glycol, a compound which promotes fusion of membranes. Upon continued incubation of ts1204-infected cells at the NPT, low numbers of capsids were assembled. Although these capsids all had some internal structure, they did not contain DNA. Another mutant, ts1208, which lies in the same complementation group as ts1204, penetrated cells normally at the NPT, but like ts1204, had a defect in the formation of functional capsids. Evidence presented in this paper suggests that the gene in which the ts1204 and ts1208 lesions map encodes a structural polypeptide.

Animals↗
PubMed2002

Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.

Studies were carried out to examine the mechanism of action of WAY-150138, a member of a novel group of thiourea compounds recently shown to inhibit replication of herpes simplex virus type 1 (HSV-1). Previous studies have shown that the drug acts by preventing DNA encapsidation and that resistant mutants map to U(L)6, the gene encoding the protein subunit of the portal complex through which DNA enters the capsid. We tested the idea that WAY-150138 acts by preventing the incorporation of DNA-packaging proteins into capsids as they are assembled. Capsids were isolated from HSV-1-infected, drug-treated cells and examined by Western immunoblotting for the presence of two packaging proteins, the portal subunit (U(L)6) and a candidate terminase subunit (U(L)15). The results showed that both proteins were depleted in the capsids, suggesting that WAY-150138 antagonizes DNA encapsidation by depriving capsids of packaging proteins during the assembly process.

Animals↗
PubMed2002

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids.

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.

Anilino Naphthalenesulfonates↗