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A virus capsid is constructed from many copies of the same protein(s). Molecular recognition is central to capsid assembly. The capsid protein must polymerize in order to create a three-dimensional protein polymer. More than structure is required to understand this self-assembly reaction: one must understand how the pieces come together in solution.
The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.
Influenza virus assembly and morphogenesis require transport of viral components to the assembly site at the apical plasma membrane of polarized epithelial cells and interaction among the viral components. In this report we have discussed the apical determinants present in the transmembrane domain (TMD) of influenza virus hemagglutinin (HA) and neuraminidase (NA), and the interaction of M1 with influenza virus HA and NA. Earlier studies have shown that the NA and HA TMDs possess determinant(s) for apical sorting and raft-association (Kundu et al., 1996. J. Virol 70, 6508-6515; Lin et al., 1998. J. Cell Biol. 142, 51-57). Analysis of chimeric constructs between NA and TR (human transferring receptor) TMDs and the mutations in the NA and HA TMD sequences showed that the COOH terminus of the NA TMD and NH(2) terminus of the HA TMD encompassing the exoplasmic leaflet of the lipid bilayers were significantly involved in lipid raft-association and that apical determinants were not discrete sequences but rather dispersed within the TMD of HA and NA. These analyses also showed that although both signals for apical sorting and raft-association resided in the NA TMD, they were not identical and varied independently. Interactions of M1 protein with HA or NA, the influenza virus envelope glycoproteins, were investigated by TX-100 detergent treatment of membrane fractions and floatation in sucrose gradients. Results from these analyses showed that the interaction of M1 with mature HA and NA, which associated with the detergent-resistant lipid rafts caused an increased detergent-resistance of the membrane-bound M1 and that M1 interacted with HA and NA both in influenza virus-infected cells as well as in recombinant vaccinia virus-infected cells coexpressing M1 with HA and/or NA. Furthermore, both the cytoplasmic tail and the TMD of HA caused an increased detergent-resistance of the membrane-bound M1 supporting their interaction with M1. Immunofluorescence analysis by confocal microscopy also showed colocalization supporting the interaction of M1 with HA and NA at the cell surface and during exocytic transport both in influenza virus-infected cells as well as in coexpressing cells.
The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.
African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249-278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.
Monensin inhibits the intracellular transport of the glycoproteins of Newcastle disease virus between cis and trans Golgi stacks of infected BHK cells, as evidenced by its effect upon their post-translational modifications such as fatty acid acylation, glycosylation and proteolytic cleavage. Thus the drug has markedly altered the subcellular distribution of the glycoproteins so that they accumulate in the internal smooth membranes but are virtually absent in the plasma membrane. These glycoproteins that accumulated in intracellular membranes have a cytoplasmic domain susceptible to protease digestion and thus are transmembranous. Under such conditions, the behavior of M protein, which plays a crucial role in virus assembly (Y. Nagai et al., 1976, Virology 69, 523-538), has been analyzed. It has been found that the M protein can neither associate with the internal membranes nor bind to the plasma membrane. Thus no virus budding has been observed, either at the plasma membranes or at internal membranes. These results substantiate the view that the interaction between M and glycoproteins is of great importance for virus assembly and suggest further that this interaction is possibly only when the glycoproteins have been incorporated into the plasma membrane.
Virus capsids assemble through the repeated interaction of well-defined protein subunits in a highly specific process. Basic research into the mechanism of protein polymerisation and virus assembly suggest that inhibition of the protein-protein interactions necessary for assembly is a valid therapeutic strategy. Computer models of virus-capsid assembly have located vulnerable stages in assembly, and small-molecule inhibitors of virus assembly have been identified. The challenge will be identifying agents that block assembly with the required specificity.
Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.
The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.
The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.
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R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J. C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981) Virology 108, 521-524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39 degrees. In 293 cells at 39 degrees, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39 degrees produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39 degrees. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39 degrees furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.
The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonas fragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.
The gene coding for the small (S) envelope protein of hepatitis B virus was mutated to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. This study was focused on a domain of the S protein that is exposed in the cytoplasm during synthesis and thereby represented a good candidate for interaction with the viral nucleocapsid during virion assembly. The mutations consisted of deletion/insertions spanning the entire cytosolic domain of S between amino acid residues 24 and 80. Although the expression of mutants clustered between residues 59 and 80 could not be obtained, we demonstrated that a large part of the cytosolic loop, from residues 29-47 and 49-59, does not contain motifs essential for production of hepatitis B virus subviral particles or HDV virions. However, deletion of residues 24-28 led to the synthesis of S protein mutant, which was competent for secretion of subviral particles but deficient for production of HDV. We concluded that the sequence between Arg-24 and Ile-28 located at the carboxyl boundary of the transmembrane signal I for S contains residue or residues important for HDV particle assembly.
Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670-7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.
The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.
Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.
Capsids of spherical viruses may be constructed from hundreds or thousands of copies of the major capsid protein(s). These assembly reactions are poorly understood. Here we consider the predicted behavior for assembly where the component reactions have weak association energy and are reversible and compare them to essentially irreversible reactions. The comparisons are based on mass action calculations and the behavior predicted from kinetic simulations where assembly is described as a cascade of low order reactions. Reversible reactions are characterized by a pseudo-critical concentration, whereas irreversible reactions consume all free subunits. Irreversible reactions are more susceptible to kinetic traps comprised of numerous small intermediates. In the case where only the ultimate step is irreversible, very low concentrations of intermediates slow the completion of the reaction so that overall it closely matches the predictions for the reversible reactions that make up the majority of the cascade. Data in the literature strongly support the hypothesis that most viruses are held together by many weak interactions.