Role of M13 gene 1 protein in filamentous virus assembly.
The M13 gene 1 protein is needed continuously for virus production but is not needed directly for the proteolytic conversion of procoat to coat.
Capsid↗
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Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.
Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.
Bacteriophage P22↗
[The effect of envelope protein mutation on hepatitis B virus assemble].
OBJECTIVE: To investigate the effect of envelope protein mutation on HBV assembly. METHODS: The envelope protein mutated vectors were constructed by the molecular clone in vitro, and then transfected transiently in the cell HepG2. The expression and secretion of S protein was assay by ELISA. HBV DNA was quantitatively evaluated by PCR. After co-transfection with pHBV-mS1S and adwR9 the DNA was quantitatively evaluated by PCR. RESULTS: There was no significant difference in expression and secretion of S protein assayed by ELISA in the cytoplasm and supernatant among pHBV-mS1, pHBV-mS, pHBV-mS1S and the wild HBV adwR9 plasmid. The DNA detected by real-time fluorescence quantitative PCR from those the cytoplasm of mutants was higher than that from the wild HBV adwR9 cytoplasm, especially from the cytoplasm of pHBV-mS1S plasmid. However, the DNA detected by real-time fluorescence quantitative PCR from the supernatant of those mutants was lower than that from the wild HBV adwR9 supernatant, especially from pHBV-mS1S. The DNA detected by real-time fluorescence quantitative PCR from the supernatant co-transfected with pHBV-mS1S and adwR9 was lower than that from the supernatant co-transfected with pcDNA3 and adwR9. CONCLUSION: There was no effect of envelope protein mutation on the expression and secretion of S protein. Envelope protein mutation could interfere the assembly of HBV particle and cause reduction of secretion of HBV.
Cell Line, Tumor↗
Role of murine leukemia virus nucleocapsid protein in virus assembly.
The retroviral nucleocapsid protein (NC) originates by cleavage of the Gag polyprotein. It is highly basic and contains one or two zinc fingers. Mutations in either the basic residues or the zinc fingers can affect several events of the virus life cycle. They frequently prevent the specific packaging of the viral RNA, affect reverse transcription, and impair virion assembly. In this work, we explore the role of NC in murine leukemia virus (MLV) particle assembly and release. A panel of NC mutants, including mutants of the zinc finger and of a basic region, as well as truncations of the NC domain of Gag, were studied. Several of these mutations dramatically reduce the release of virus particles. A mutant completely lacking the NC domain is apparently incapable of assembling into particles, although its Gag protein is still targeted to the plasma membrane. By electron microscopy on thin sections of virus-producing cells, we observed that some NC mutants exhibit various stages of budding defects at the plasma membrane and have aberrant particle morphology; electron micrographs of cells expressing some of these mutants are strikingly similar to those of cells expressing "late-domain" mutants. However, the defects of NC mutants with respect to virus release and infectivity could be complemented by an MLV lacking the p12 domain. Therefore, the functions of NC in virus budding and infectivity are completely distinct from viral late-domain function.
Amino Acid Sequence↗
The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor.
Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-A-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature- and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy.
Capsid Proteins↗
Rotavirus protein expression is important for virus assembly and pathogenesis.
Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.
Animals↗
Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.
During the late phase of HIV type 1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to the plasma membrane of most hematopoietic cell types, where they colocalize at lipid rafts and assemble into immature virions. Membrane binding is mediated by the matrix (MA) domain of Gag, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Although exposure is known to promote membrane binding, the mechanism by which Gag is targeted to specific membranes has yet to be established. Recent studies have shown that phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P(2)], a factor that regulates localization of cellular proteins to the plasma membrane, also regulates Gag localization and assembly. Here we show that PI(4,5)P(2) binds directly to HIV-1 MA, inducing a conformational change that triggers myristate exposure. Related phosphatidylinositides PI, PI(3)P, PI(4)P, PI(5)P, and PI(3,5)P(2) do not bind MA with significant affinity or trigger myristate exposure. Structural studies reveal that PI(4,5)P(2) adopts an "extended lipid" conformation, in which the inositol head group and 2'-fatty acid chain bind to a hydrophobic cleft, and the 1'-fatty acid and exposed myristyl group bracket a conserved basic surface patch previously implicated in membrane binding. Our findings indicate that PI(4,5)P(2) acts as both a trigger of the myristyl switch and a membrane anchor and suggest a potential mechanism for targeting Gag to membrane rafts.
Allosteric Site↗
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↗
Varicella-zoster virus assembly protein p32/p36 is present in DNA-containing as well as immature capsids.
Varicella-zoster virus (VZV) produces a group of nucleocapsid proteins (the p32/p36 nucleoprotein complex) which are the VZV analogues of the herpes simplex virus type 1 (HSV-1) and cytomegalovirus (CMV) assembly proteins. There are multiple components in the VZV p32/p36 complex, with major proteins of 32 and 36 kDa and minor proteins of 34 and 38 kDa. In HSV-1 the assembly proteins have been shown to be present in immature (B) capsids, but are removed prior to the formation of mature (C) capsids containing the viral DNA genome. Our work has shown that VZV produces capsids corresponding to the B and C forms. However, in contrast to HSV-1, VZV also produces "B/C" capsids that appear to contain both the assembly proteins and the viral DNA genome. Possible mechanisms for this are discussed. In addition, it was shown that VZV capsids appear to lack the 36 and 38 kDa proteins, and based on this observation we suggest that these may represent unprocessed forms of the assembly protein. In both HSV and CMV, a much larger, cross-reactive protein has been identified as the full-length product of the gene coding for the assembly protein. The homologous VZV gene (ORF 33) theoretically has the capacity to produce a 66 kDa protein. However, no such protein is readily apparent in VZV-infected cells. The presence of an immunoreactive 64 kDa protein was demonstrated in purified VZV capsids which may represent the full-length ORF 33 protein.
Antibodies, Monoclonal↗
Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins.
Among the three related L, M, and S envelope proteins of the hepatitis B virus (HBV), the L and S polypeptides are required for virion production. Whereas the pivotal function of the pre-S region of L in nucleocapsid envelopment has been established, the contribution of its S domain and the S protein is less clear. In this study, we evaluated the role of the cytosolic S loop, common to L and S, in HBV assembly by performing mutagenesis experiments. To distinguish between the effect of the mutations on either envelope or virion formation, we investigated the ability of the mutants to assemble into secretable subviral empty envelopes and to replace the wild-type proteins in virion maturation, respectively. Virion production was found to be blocked by each of the secretion-competent deletion and substitution mutants SDelta35-39, SDelta40-46, SDelta50-56, and Svarsigma56-59, while an insertion within the loop is tolerated. Surprisingly, single mutations of the arginines terminating the loop had an opposite effect: while a conservative exchange of Arg-73 still allowed virion formation, the same mutation of Arg-79 did not. The critical sequences and/or structural requirements of the cytosolic S loop involved in nucleocapsid envelopment primarily act in the S background. These findings can be related to a model for a synergistical function of both L and S proteins in HBV morphogenesis.
Amino Acid Sequence↗
Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly.
In addition to the major surface (S) protein, the envelope of the duck hepatitis B virus (DHBV) contains a related presurface (preS) protein whose N-terminus bears a covalently attached myristate group. We have explored the functional significance of this modification by examining the replicative potential of a mutant viral genome whose myristylation signal has been inactivated. Following transfection into permissive hepatoma cells, the mutant expresses an unmyristylated preS protein of normal size, immunoreactivity and stability. Cytoplasmic cores containing viral DNA are synthesized, and Dane particles are assembled and exported into the medium. However, the mutant is noninfectious when inoculated into susceptible ducklings. We conclude that myristylation of preS proteins is essential for hepadnaviral infectivity but not for viral assembly; myristylation is most likely required for an early step of the life cycle involving the entry or uncoating of virus particles.
Amino Acid Sequence↗
Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly.
Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was studied. The half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H was approximately 3 h for the recombinant feline infectious peritonitis virus S protein. The S protein in feline infectious peritonitis virus-infected cells was found to have a half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H of approximately 1 h. This difference can be explained by the fact that coronavirus budding takes place at intracellular membranes and that the oligosaccharides of the spike protein are modified after budding. Apparently, spike protein incorporated into budded virions is transported faster through the Golgi apparatus than is spike protein alone. These findings provide new insights into the mechanism of coronavirus budding and are discussed in relation to current models of intracellular transport and sorting of proteins.
Animals↗
The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.
The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.
Base Sequence↗
Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22.
A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.
Bacteriophage P22↗
Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles.
The production of infectious particles by human immunodeficiency virus type 1 is dependent on the accurate cleavage of its Gag and Gag/Pol precursors by a virally encoded protease. In the absence of protease activity, morphologically abnormal particles which are noninfectious are formed. Recently, inhibitors of the protease of human immunodeficiency virus type 1 have been developed as potential therapeutic agents. We have examined the basis for the loss of infectivity at the limiting inhibitor concentrations that are likely to be achieved in clinical settings. We found that subtle defects in processing are correlated with profound deficits in infectivity. Further, we correlated this partially disrupted processing with an altered virion morphology. These data suggest that accurate and complete processing is essential to the formation of infectious, morphologically normal virions and that the pathway by which these precursors are processed and assembled is sensitive to partial inhibition of the protease by an inhibitor disproportionate to the effect of the inhibitor on the viral protease itself.
Cells, Cultured↗
Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly.
We have used the method of chemical crosslinking in order to determine the spatial interactions between components of Rous sarcoma virus. A high molecular weight complex formed by crosslinking has been isolated by ultracentrifugation on sucrose density gradients containing 0.1% (w/v) sodium dodecyl sulphate. This complex is composed of the two viral glycoproteins gp85 and gp35, the gag protein p19, and the viral RNA. Two types of bonding are important for the formation and stability of the complex: first, native disulphide bonds between gp85 and gp35 and between individual p19 molecules; and second, hetero-crosslinking between gp35 and p19 as well as homo-crosslinking between p19. Although viral RNA is quantitatively present in the complex, experiments with RNase treatment show that it is not essential for its formation or stability. A small amount of lipid is present in the complex and appears to be crosslinked to p19. In vitro-labelling of purified virus with the lipophilic photoactivatable reagent [125I] iodonaphthylazide resulted in the labelling of gp35 and p19/23. In vivo-labelling of virus with [3H]palmitate resulted in only gp35 becoming labelled. These results substantiate the membrane association of these proteins. The significance of the interactions in the high molecular weight complex for the stability of the virus and, by implication, the role which they may play in viral assembly are discussed.
Avian Sarcoma Viruses↗
Differences in pressure stability of the three components of cowpea mosaic virus: implications for virus assembly and disassembly.
A comparison of pressure stability of empty capsids and ribonucleoprotein particles of cowpea mosaic virus (CPMV) is presented. A combination of high pressure and subdenaturing concentrations of urea was utilized to promote dissociation and denaturation. We found that RNA plays an important role in stabilizing the particles as well as in conferring reversibility to the pressure-induced denaturation. Dissociation and denaturation of the top component (empty capsid) was observed at 2.5 kbar and in the presence of 2.5 M urea. The pressure-dissociated state of the capsid protein had the characteristics of a denatured conformation as suggested by fluorescence spectra, lifetime of tryptophans, and binding of bis-ANS. The properties of the dissociated capsid protein were more similar to those of a molten-globule conformation, different from the more drastically unfolded state obtained using high concentrations of urea. Whereas the fluorescence of bis-ANS increased for the pressure-dissociated protein (1.5 M urea and 2.5 kbar), it decreased for the virus denatured by 6.0 M urea. Middle and bottom components underwent less than 50% change in center of spectral mass at 2.5 kbar and 2.5 M urea. The particles containing RNA could be fully affected by pressures of 2.5 kbar--as measured by the spectral shift--only in the presence of 5.0 M urea. RNA-containing capsids denatured by pressure did not bind bis-ANS, suggesting that the capsid protein continues to be bound to the RNA after the protein-protein contacts are broken by pressure. Reassembly of the nucleoprotein particles was obtained after decompression, reinforcing the idea that proteins had not dissociated from RNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Capsid↗