Organization of the Sindbis virus nucleocapsid as revealed by bifunctional cross-linking agents

Purified Sindbis virus nucleocapsids were reacted with a variety of bifunctional protein-specific cross-linking agents. The products were analyzed in concentration-gradient polyacrylamide gels and amounts of various products determined. These studies indicated that available lysine residues within adjacent capsid proteins in purified intact nucleocapsids are separated by 6 A. The capsid proteins in intact nucleocapsids are cross-linked in a pattern predicted for discrete monomeric entities, rather than in dimeric or trimeric aggregates. Purified, soluble capsid protein exists in a conformation that differs from the arrangement of protein within nucleocapsids. These conformational differences suggest that topological changes may occur in the capsid protein during virus maturation. Cross-linked nucleocapsids that were treated with RNases resulted in the generation of RNA-free protein shells that retained hexagonal morphology, indicating that, together, the RNA and protein form the outer surface of the nucleocapsid. These data are used to produce a model of the Sindbis virus nucleocapsid in which the proteins are arranged quasi-equivalently in a T = 4 icosahedral shell.     

Is Sindbis a simple picornavirus with an envelope?

A three-dimensional image reconstruction was performed from cryo-electron micrographs of isolated Sindbis (SNV) nucleocapsids. The isolated capsid is a smooth but fenestrated T = 3 structure. Comparison with the nucleocapsid seen within the whole virion indicated that the structure resembles the swollen forms which some non-enveloped viruses adopt after removal of divalent cations. A sensitive comparison method was used to align SNV capsid protein sequences with those of picornavirus vp3 capsid proteins whose high resolution structures display an eight-stranded beta-barrel fold found in many icosahedral viruses. The alignment predicted a similar folding for the Sindbis protein which juxtaposes several sets of residues known to be essential for its serine proteolytic activity. These results suggest that the capsid proteins of the enveloped alphaviruses and the non-enveloped picornaviruses may have arisen through divergent evolution from a simple, vp3-like ancestor.     

Role of ribosomes in Semliki Forest virus nucleocapsid uncoating.

The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.     

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.     

The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability

The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg(2+) ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.     

Segmented genome and nucleocapsid of La Crosse virus.

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Sindbis virus nucleocapsid assembly: RNA folding promotes capsid protein dimerization

In Sindbis virus, initiation of nucleocapsid core assembly begins with recognition of the encapsidation signal of the viral RNA genome by capsid protein. This nucleation event drives the recruitment of additional capsid proteins to fully encapsidate the genome, generating an icosahedral nucleocapsid core. The encapsidation signal of the Sindbis virus genomic RNA has previously been localized to a 132-nucleotide region of the genome within the coding region of the nsP1 protein, and the RNA-binding activity of the capsid was previously mapped to a central region of the capsid protein. It is unknown how capsid protein binding to encapsidation signal leads to ordered oligomerization of capsid protein and nucleocapsid core assembly. To address this question, we have developed a mobility shift assay to study this interaction. We have characterized a 32 amino acid peptide capable of recognizing the Sindbis virus encapsidation signal RNA. Using this peptide, we were able to observe a conformational change in the RNA induced by capsid protein binding. Binding is tight (K(d)(app) = 12 nM), and results in dimerization of the capsid peptide. Mutational analysis reveals that although almost every predicted secondary structure within the encapsidation signal is required for efficient protein binding, the identities of the bases within the helices and hairpin turns of the RNA do not need to be maintained. In contrast, two purine-rich loops are essential for binding. From these data, we have developed a model in which the encapsidation signal RNA adopts a highly folded structure and this folding process directs early events in nucleocapsid assembly.     

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.     

Structural Transitions in Southern Bean Mosaic Virus and their Correlation with Infectivity and Ribonuclease Sensitivity

Abstract Southern bean mosaic virus (SBMV) virions swell when the capsid-associated divalent cations are removed with EDTA at pH 7.5, resulting in an approximate 99% decline in the specific infectivity. Recompacting swollen virions either with divalent cations at pH 7.5, or by lowering the pH to 5.0 (in the absence or in presence of divalent cations) fails to restore complete infectivity. In contrast to swollen SBMV, RNA within the recompacted virions is fully protected from ribonuclease attack. Removing divalent cations with EDTA at pH 5.0 causes no infectivity loss or conformational change. These results indicate that if SBMV conformation is altered once then an irreversible loss in the infectivity occurs and the divalent cations play no role per se , in the infection process. Furthermore, observations based upon the sedimentation behaviour of ribonuclease-treated SBMV indicate that RNA must be physically intact for capsid recompaction to occur. Obviously, structural rearrangements at the capsidsurface (e.g., regeneration of intersubunit interactions) and at the virion interior (i.e., RNA-protein linkages) are involved collectively in conferring conformational stability to the recompacted SBMV.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Role of sindbis virus capsid protein region II in nucleocapsid core assembly and encapsidation of genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.     

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.     

Rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation

During virus assembly, the capsid proteins of RNA viruses bind to genomic RNA to form nucleocapsids. However, it is now evident that capsid proteins have additional functions that are unrelated to nucleocapsid formation. Specifically, their interactions with cellular proteins may influence signaling pathways or other events that affect virus replication. Here we report that the rubella virus (RV) capsid protein binds to poly(A)-binding protein (PABP), a host cell protein that enhances translational efficiency by circularizing mRNAs. Infection of cells with RV resulted in marked increases in the levels of PABP, much of which colocalized with capsid in the cytoplasm. Mapping studies revealed that capsid binds to the C-terminal half of PABP, which interestingly is the region that interacts with other translation regulators, including PABP-interacting protein 1 (Paip1) and Paip2. The addition of capsid to in vitro translation reaction mixtures inhibited protein synthesis in a dose-dependent manner; however, the capsid block was alleviated by excess PABP, indicating that inhibition of translation occurs through a stoichiometric mechanism. To our knowledge, this is the first report of a viral protein that inhibits protein translation by sequestration of PABP. We hypothesize that capsid-dependent inhibition of translation may facilitate the switch from viral translation to packaging RNA into nucleocapsids.     

Analyses of phosphorylation events in the rubella virus capsid protein: role in early replication events

The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

cis-acting genomic elements and trans-acting proteins involved in the assembly of RNA viruses

There is now considerable evidence that a specific site (or sites) in the genome of an RNA virus interacts with a viral protein to initiate the assembly of the virus ribonucleoprotein or nucleocapsid. We describe the progress that has been made in defining these elements for a number of different viruses: the togavirus, Sindbis virus; the coronavirus, mouse hepatitis virus; influenza A virus; several retroviruses; and the hepadnavirus, hepatitis B virus. The importance of cis-acting elements in packaging has been established for all of these viruses. For Sindbis virus, specificity in the binding of the RNA element to a region of the viral capsid protein in vitro has also been demonstrated.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.