The phylogenetic structure of the cluster of tobamovirus species serologically related to ribgrass mosaic virus (RMV) and the sequence of streptocarpus flower break virus (SFBV).

Ribgrass mosaic virus (RMV), turnip vein-clearing virus (TVCV) and Youcai mosaic virus (YoMV; formerly designated as oilseed rape mosaic virus; ORMV) belong to the genus Tobamovirus and are arranged in one out of three subgroups because of their common host range, serological cross-reactivity and amino acid composition of their coat proteins. The recently defined species Wasabi mottle virus (WMoV) is closely related to the same subgroup. The distinction of the four species is difficult and the lack of sequence information of a wide range of isolates has led to an unclear nomenclature. To clarify this situation we sequenced the coat protein genes from 18 isolates which were serologically related to members of the species of this cluster. The size of the coat protein was conserved with the exception of one isolate which revealed an N-terminal extension due to the mutation of three stop-codons. Phylogenetic analysis of these CP ORFs resulted in a tree with three clusters each containing at least one of the approved species RMV, TVCV and 1ptYoMV/WMoV in which our isolates were distributed. The tree was congruent and did support the present taxonomic status of species within this subgroup. For practical purpose we developed a subgroup 3 specific primer pair and a species differentiating restriction fragment length polymorphism (RFLP). Sequencing of the genome of Streptocarpus flower break virus (SFBV) which is serologically distantly related to the subgroup 3 viruses revealed a distinct genome organization. Therefore we propose that this virus should be regarded as a member of a species not belonging to any of the subgroups so far established.

Pelargonium necrotic spot virus: a new member of the genus Tombusvirus.

A virus isolate from Pelargonium spp., provisionally designated UPEV (unknown pelargonium virus), had isometric particles 31-33 nm in diameter, with a granular surface structure similar to that of viruses in three genera of family Tombusviridae. Immunoelectron microscopy proved that UPEV was serologically distinct from all examined morphologically similar members of the family Tombusviridae. The induced cytopathology was characterized by large cytoplasmic virion aggregates and the formation of multivesicular bodies derived from mitochondria. Analysis of the complete ssRNA genome sequence revealed four open reading frames (ORFs) arranged like those of viruses in the genera Tombusvirus and Aureusvirus. Sequence comparisons indicated that three of the four ORFs had a high identity (52-97% identical amino acids) with the respective ORFs of tombusvirus species, especially with Carnation Italian ringspot virus, but not with those of viruses in other genera in Tombusviridae. On the contrary, UPEV coat protein had a low indentity (36-53% identical amino acids) with that of the aureusvirus Pothos latent virus. The data suggested that UPEV originated in a recombination event between a tombus- and an aureusvirus. According to its original host and symptom expression we proposed the new virus be named Pelargonium necrotic spot virus (PeNSV) and classified it as a distinct and new species in the genus Tombusvirus.

An unusual large intergenic region in the S-RNA of a Bulgarian tomato spotted wilt virus isolate.

The complete S-RNA sequences of four Bulgarian and one German isolate of tomato spotted wilt tospovirus were determined. All isolates show a high conservation in their N proteins, while the NSs proteins and the intergenic regions (IGR) are more variable. The Bulgarian isolate 10HK96 has the largest S-RNA (3364 nucleotides) among tomato spotted wilt viruses reported so far. The enlargement is based on an insertion of 365 nts in the IGR that may have resulted from stuttering of the viral polymerase or non-homologous recombination. This insertion is present in the N protein gene subgenomic messenger, upstream of a proposed transcription termination signal.

Variability of the N-protein and the intergenic region of the S RNA of tomato spotted wilt tospovirus (TSWV).

The genetic heterogeneity of the N protein gene and the intergenic region (IGR) of the S RNA from tomato spotted wilt virus (TSWV) isolates, collected in Bulgaria, were compared with isolates from other parts of the world. The results substantiated the highly conserved nature of the N protein. Twenty six independent sequences revealed only seven variable amino acid positions, common to all isolates. The type of amino acids present in these positions seems to be independent of the geographical origin. In contrast to the structural N protein, comparisons of the related IGR-sequences led to clusters correlated with the geographical origin of the isolates. Although the overall sequence homology in the IGRs was much lower than for the N proteins, three conserved parts within this region were identified. The outstanding part was a central area of 31 nucleotides with a significantly increased GC-content. This was located in both viral- and viral-complement RNA at structures with similar foldings, which led to the assumption that this stabilised structure, rather than a sequence motive, might serve as a transcription terminator during the synthesis of the two mRNAs from the ambisense segments.

Identification of a functionally important amino acid residue near to the amino-terminus of the human immunodeficiency virus type 1 Vif protein.

Previous studies have reported the importance of residues in the central and carboxy-terminal regions of HIV-1 Vif as being important for a Vif+ phenotype. Mutants in a residue (Trp21) near to the amino-terminus of Vif, which is conserved between HIV-1, HIV-2, and SIVmac, were generated in the permissive C8166 cell line, and their Vif phenotype determined by monitoring their growth on the nonpermissive H9 cell line. Of seven variants at position 21 of Vif only phenylalanine substituted successfully for Trp21. The expression of the mutant Vif proteins was shown to be similar to wildtype levels, in C8166 cells, in all cases except one. These data identify Trp21 as an amino-terminal residue which is important to Vif function, in allowing growth of virus in nonpermissive H9 cells, and suggest that there is limited scope for variation at this position of HIV-1 Vif.

Crystal structure of vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure-based drug design.

A comparison of the anti-rhinoviral drug binding pocket in HRV14 and HRV1A.

The three-dimensional structures of two human rhinovirus serotypes (HRV14 and HRV1A) are compared when complexed with various antiviral agents. Although these agents all bind into the same hydrophobic pocket, the exact viral-drug interactions differ. In the absence of drugs, the pocket is occupied by a fatty acid in HRV1A, but is empty in HRV14 except for two water molecules. The conformation of each drug is dependent upon the shape of the hydrophobic pocket. In HRV14 the major residues determining the shape of the binding site are Y1128, P1174 and M1224, corresponding to I1125, M1169 and I1220 in HRV1A. When there is no cofactor or a drug in the pocket, the entrance to the pocket is open. However, the entrance is closed when the pocket is occupied by a cofactor or a drug. There are relatively small conformational changes when the agents displace the natural cofactor in HRV1A. In contrast, there are much larger conformational changes on binding a drug in HRV14. These differences cause an inhibition of viral attachment in HRV14 but not in HRV1A. Binding of the drugs results in three additional interprotomer hydrogen bonds in HRV14 and one in HRV1A. These hydrogen bonds and a potential loss of flexibility upon efficient packing of the pocket may contribute to the inhibition of uncoating in both serotypes.

Structure determination of the bacteriophage phiX174.

The structure of the single-stranded DNA phage phiX174 has been determined to 3.4 A resolution. The crystal space group was P2(1) with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy. Oscillation diffraction photographs were collected using synchrotron radiation at various wavelengths. The particle orientations in the unit cell were determined with a rotation function. Because cowpea mosaic virus has a similar external envelope to phiX174, it was used as a search model to find the approximately positions of the phiX174 particles in the unit cell relative to the crystallographic symmetry axes. An initial phase set to 12 A resolution was then based on the cowpea mosaic virus atomic structure. These phases were improved by 20 cycles of real-space molecular replacement averaging. The phase information was gradually extended to 3.4 A resolution by molecular replacement electron density averaging. One reciprocal lattice point was used for each extension followed by four cycles of averaging. The unusual particle capsid, with its 12 pentameric spikes, required the careful determination of a precise molecular envelope. This was redetermined at regular intervals, as was the particle center. The resultant electron density map was readily interpreted in terms of the F, G and J polypeptides in the capsid. A difference electron density map between full and partially empty particles showed some ordered DNA structure.

The three-dimensional structure of frozen-hydrated bacteriophage phi X174.

The three-dimensional structure of bacteriophage phi X174 (phi X174) was determined to approximately 2.6 nm resolution from images of frozen-hydrated 114 S particles. The outer surface of phi X174 is characterized by several prominent features: (i) 12 mushroom-shaped caps (approximately 7.1 nm wide x 3.8 nm high) are situated at each of the vertices of the icosahedral virion and extend to a maximum radius of 16.8 nm; (ii) a "collar" of density surrounds the base of each apical cap; and (iii) 20 conical protrusions (approximately 2.3 nm high) lie along the three-fold symmetry axes. The caps have a pentagonal morphology composed of five globular "subunits" and appear to be loosely connected to the underlying capsid. The distribution of the four gene products present in virions (60 copies each of gpF, gpG, and gpJ, and 12 copies of gpH), and the single-stranded DNA (ssDNA) genome cannot be directly discerned in the reconstructed density map, although plausible assignments can be made on the basis of solvent-excluded volume estimates and previous biochemical data. Thus, gpG accounts for most of the mass in the caps; gpH, a presumed cap protein, cannot be identified in part due to the symmetry-averaging procedures, but may be partially located within the interior of the capsid; and gpF and gpJ make up the remainder of the capsid. The genome appears to be less densely packaged inside the capsid compared to many dsDNA viruses whose nucleic acid is arranged in a liquid-crystalline state.

Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure,

Preliminary investigation of the phage phi X174 crystal structure.

Crystals of the single-stranded DNA bacteriophage phi X174 have been grown. They have a monoclinic unit cell with space group P2(1), unit cell dimensions of a = 306.0 (+/- 0.2) A, b = 361.1 (+/- 0.2) A, c = 299.7 (+/- 0.2 degrees) A, beta = 92.91 degrees (+/- 0.02 degrees) and diffract to at least 2.7 A resolution. There are two virus particles per unit cell. Packing considerations show that the mean diameter of the virus particles is 280 A. The virus separates into two bands in a sucrose gradient. The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles. Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes.

Recovery of structurally intact and infectious poliovirus type 1 from HeLa cells during receptor-mediated endocytosis.

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.

Inhibition of poliovirus uncoating by disoxaril (WIN 51711).

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.

Neutralization of poliovirus by polyclonal antibodies requires binding of a single IgG molecule per virion.

Neutralization of poliovirus type 1 was studied using radioactively labelled polyclonal IgG. With nonsaturating antibody concentrations various virus-antibody complexes were produced which were isolated by sucrose gradient centrifugation and identified by electron microscopy as virus monomers, dimers, trimers, tetramers and pentamers. The neutralization rate (n.r.) of each of the virus-antibody complexes relative to non-neutralized virus and the stoichiometry have been estimated. The monomer fraction showed that about every fifth virion was associated with one IgG molecule and neutralized. The antibody was bivalently attached. The majority of virus particles formed aggregates of different sizes, which were cross-linked by antibodies. The following neutralization rates and ratios of IgG to virus (IgG/V) were determined for the oligomers: dimers, 59.2 per cent n.r. and 0.55 IgG/V; trimers, 67.3 per cent n.r. and 0.66 IgG/V; tetramers, 79.0 per cent n.r. and 0.75 IgG/V; pentamers, 86.3 per cent n.r. and 0.98 IgG/V. Two different mechanisms of neutralization are proposed: i) an antibody-mediated mechanism specifically inhibits infectivity of the monomer virus-antibody complexes and ii) reduction of infectivity of oligomer virus-antibody complexes is caused simply by reduction of the actual number of infectious units. Immunoprecipitation of the denatured capsid proteins showed that only VP 1 was recognized by the polyclonal IgGs.

Entry of poliovirus type 1 and Mouse Elberfeld (ME) virus into HEp-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating.

Poliovirus type 1 appeared from electron microscope studies to enter HEp-2 cells by receptor-mediated endocytosis. On adsorption the virus was evenly distributed over the cell surface, with some preference for the microvilli and their bases. Invagination of the cell surface membrane with the attached virus commenced at coated pits and led to the formation of virus-containing coated vesicles in the cytoplasm. These coated vesicles fused with intracellular vesicles to form endosomes. When cells infected with poliovirus or Mouse Elberfeld virus were treated with the weak bases chloroquine, NH4Cl or the ionophore monensin to raise the intraendosomal and intralysosomal pH above 6, virus-directed macromolecular synthesis and production of progeny were prevented. These results suggest that the virus genomes are released to the cytoplasm via endosomes and/or lysosomes by a pH-dependent process.

Dense particles and slow sedimenting particles produced by ultraviolet irradiation of poliovirus.

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.