Virus Capsid Structures Research Articles

Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs.

Plasmids containing the foot-and-mouth disease virus structural protein precursor (P1) and 3C protease genes or the P1 gene alone were expressed in Escherichia coli. A recombinant baculovirus containing the P1 gene was also generated and expressed in Spodoptera frugiperda cells. Expression of the P1 and 3C genes in E. coli resulted in efficient synthesis and processing of the structural protein precursor and assembly into 70S empty capsids. This material reacted with neutralizing monoclonal antibodies which recognize only conformational epitopes and elicited a significant neutralizing antibody response in vaccinated guinea pigs. Expression of the P1 gene in E. coli resulted in synthesis of an insoluble product, whereas in insect cells infected with the recombinant baculovirus a soluble product was synthesized. Both soluble and insoluble P1 reacted with a 12S-specific monoclonal antibody, but only soluble P1 elicited a neutralizing antibody response in guinea pigs.

Nucleotide Sequences of Coat Protein Genes for Three Isolates of Barley Yellow Dwarf Virus and their Relationships to Other Luteovirus Coat Protein Sequences

Barley yellow dwarf virus (BYDV) can be separated into two groups based on, among other criteria, serological relationships that are presumably governed by the viral capsid structure. Nucleotide sequences for the coding regions of coat proteins of approximately 22 K were identified for the MAV-PS1, P-PAV (group 1) and NY-RPV (group 2) isolates of BYDV. The MAV-PS1 and P-PAV coat protein sequences shared 71% deduced amino acid similarity whereas that of the NY-RPV isolate shared no more than 51% similarity with either the MAV-PS1 or the P-PAV sequence. Other comparisons showed that these and other BYDV coat protein sequences examined to date share a high degree of identity with those identified from other luteoviruses. Among luteovirus coat protein sequences in general, several highly conserved domains were identified whereas other domains differentiate MAV-PS1 and PAV isolates from NY-RPV and other luteoviruses. Sequence similarities and differences among BYDV coat proteins (approx. 22K) are consistent with the serological relationships exhibited by these viruses. Amino acid sequence comparisons between BYDV isolates that share common aphid vectors indicate that it is unlikely that these coat proteins are involved in aphid specificity.

Hepatitis A Virus Aggregation in Suspensions of Purified Virus

Virus capsid structure as determined by x-ray crystallography or by elec imaging of virus particles in thefrozen hydrated state has received con interest during the past decade. Specific functional proteins were localized recently on the Rotavirus capsid by using cryo-electron microscopy, indicating the potential for morphological studies that rel directly to capsid protein function.Most cryo-electron microscopy studies have involved investigation of vir were 40 nm or greater in diameter. Smaller viruses may provide a greate practical difficulty in their preparation and imaging.Highly purified virus for study by x-ray crystallographic studies or ele microscopic evaluation of frozen hydrated particles requires stringent c particle aggregation. Preparations for x-ray crystallography should agg an orderly manner to form arrays and ordered crystals of sufficient size Preparations for cryo-electron microscopy are more desirable when the pa numerous but not over-lapping (Fig. 1). We have experienced uncontrolla aggregation including considerable over-lapping of hepatitis A virus (HA particles when purified preparations were stored in phosphate or tris buffers (Fig. 2).

Biochemistry and pathogenicity of echovirus 9. III. Thermosensitive mutants of echovirus 9, strain Barty, with reduced pathogenicity for newborn mice.

Different clinical isolates of echovirus 9 are known to vary strikingly with regard to pathogenicity. Prototype strain Hill and strain Barty have previously been shown to differ not only in paralytogenic potency for newborn mice but also in a number of in vitro characteristics related to virus capsid structures. A series of mutants of strain Barty, thermosensitive for replication at 40 degrees C, was isolated after mutagenization with 5-fluorouracil. For all mutants the virus dose required to paralyze 50% of the infected animals was significantly higher than of the parent strain Barty. This reduced pathogenicity was observed at normal room temperature where the baby mice had a body temperature of 32.5 degrees C, which is even below the permissive temperature for growth of the mutants. The paralytogenic potencies did not further decrease when the mice where kept at elevated room temperature and had a body temperature of 35.1 degrees C. Thus, the reduced pathogenicity is apparently not a direct consequence of thermosensitivity of growth. Biochemical and biophysical characterization indicated that at least two of the eight mutants have an alteration in capsid protein.

Biochemistry and pathogenicity of echovirus 9 II. Mutants of echovirus 9, strain Hill, with altered capsid surface properties

The two echovirus 9 strains Hill and Barty have been shown previously not only to differ in pathogenicity for newborn mice but also in a number of in vitro characteristics which depend on viral capsid structure. Three spontaneously occurring mutants of the mouse-apathogenic echovirus 9 prototype strain Hill being resistant to an inhibitor of plaque formation present in agar were isolated and compared biochemically and biophysically to their parent strain and to the mouse-pathogenic echovirus 9 strain Barty, which is resistant to this inhibitor. The mutants differ from their apathogenic parent strain Hill in most of the same in vitro characteristics as strain Barty, namely adsorption to cells in culture, sedimentation behavior in low salt sucrose gradients, distribution of mutant virus particles in isoelectric focusing, and antigenic determinants inducing neutralizing antibodies. For two of the three mutants, Ag2 and Ag3, evidence was obtained from fingerprinting that they differ from their parent strain Hill in VP1; thus, the observed in vitro properties may be caused by the change in this capsid protein. All mutants, however, were found to be apathogenic for newborn mice and do not replicate in the tissues of these animals. It is concluded that the observed changes in capsid structure do not covary with virulence.

Molecular replacement method at low resolution: optimum strategy and intrinsic limitations as determined by calculations on icosahedral virus models

An extensive series of phase refinements on model structure factors has been carried out to determine the scope and limitations of real-space symmetry averaging at low resolution. These calculations were used to derive and justify the strategy used to solve the structure of polyoma virus capsids at 22.5 ,/k resolution [Rayment, Baker, Caspar & Murakami (1982). Nature (London), 295, 110-115]. The calculations showed (1) that phases from a wide variety of models can be successfully refined; (2) a low R factor (,-,10%) between the constrained and observed amplitudes corresponds to a small error between the calculated and true phases; (3) on convergence to a low R factor there is no residual bias in the final phases introduced by the initial phasing model. The model calculations were also used to determine the limitations imposed by series termination, unmeasured data, random errors in the data and the role of the molecular envelope on the phase refinement.