Structure of the black beetle virus genome and its functional implications

The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5' non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3' proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.     

Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

Studies of virus structure by laser-Raman spectroscopy. Turnip yellow mosaic virus and capsids.

Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion. The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid. Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form. Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules. Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion. The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV.     

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.     

Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus

Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants.     

A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.     

Bovine viral diarrhea virus core is an intrinsically disordered protein that binds RNA

Pestiviruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and close relatives of hepatitis C virus. Pestivirus particles are composed of an RNA genome, a host-derived lipid envelope, and four virion-encoded structural proteins, core (C), E^rns, E1, and E2. Core is a small, highly basic polypeptide that is processed by three enzymatic cleavages before its incorporation into virions. Little is known about its biological properties or its role in virion assembly and structure. We have purified BVDV core protein and characterized it biochemically. We have determined that the processed form of core lacks significant secondary structure and is instead intrinsically disordered. Consistent with its highly basic sequence, we observed that core binds to RNA, although with low affinity and little discernible specificity. We found that BVDV core protein was able to functionally replace the nonspecific RNA binding and condensing region of an unrelated viral capsid protein. Together these results suggest that the in vitro properties of core may reflect its mechanism of action in RNA packaging and virion morphogenesis.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.     

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

A single amino acid mutation in the carnation ringspot virus capsid protein allows virion formation but prevents systemic infection

A Carnation ringspot virus (CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser-->Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related Red clover necrotic mosaic virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses.     

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.     

Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation

Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs.     

A hydrophobic heptad repeat of the core protein of woodchuck hepatitis virus is required for capsid assembly.

The capsid particle of hepadnaviruses is assembled from its dimer precursors. However, the mechanism of the protein-protein interaction is still poorly understood. A small region in the capsid protein of woodchuck hepatitis virus (WHV) contains four hydrophobic residues, including leucine 101, leucine 108, valine 115, and phenylalanine 122, that are conserved and spaced every seventh residue in the primary sequence to form a hydrophobic heptad repeat (hhr). A hydrophobic force often plays an important role in the interaction of proteins. Therefore, to investigate the role of this region in capsid assembly, we individually changed the codons specifying these four hydrophobic amino acids to codons specifying alanine or proline. In addition, we examined the in vivo infectivity of a WHV genome bearing a naturally occurring single amino acid change (histidine 104-->proline) in the hhr region. The phenotype of each altered genome was determined in both eukaryotic and prokaryotic systems by a capsid protein assay and electron microscopic examination. We show that replacement of any one of the four hydrophobic residues with alanine did not prevent capsid assembly. However, assembled capsid particles were not detected if combinations of any two of the four residues were substituted with alanines or if the spacing of these four hydrophobic residues was changed. An individual introduction of a proline (which dramatically changes the secondary structure of proteins) into different positions of this small region also abolished capsid assembly in vitro or viral replication in vivo. These results suggested that the hhr region of the core protein of WHV was critical for capsid assembly.     

Nature and specificity of the RNA-protein interaction in the case of the tymoviruses.

Dissociation-reassociation experiments performed with turnip yellow mosaic virus in the presence of various RNAs and polynucleotides were used to investigate the degree of specificity and the contribution of the associated RNA moiety to the stability of TYMV. The results emphasize the importance of strategic cytosine residues spread along the RNA chain. Some insight into the contribution of the protein could be gained from comparison of TYMV and eggplant mosaic virus (EMV), a virus similar to TYMV although its top component contains low molecular mass RNA's able to bind various amino acids. Hydrophobic interactions between protein subunits are less important in EMV than in TYMV, and artificial capsids could be obtained from dissociated EMV coat protein. Whether the capsid is or is not the precursor of the virion in tymovirus morphogenesis is discussed.