Jeffrey W. Almond

Antigenic structure of polioviruses of serotypes 1, 2 and 3.

The antigenic sites recognized by monoclonal antibodies with neutralizing activity for the Sabin vaccine strains of poliovirus of serotypes 1, 2 and 3 have been studied by the isolation and characterization of mutants resistant to neutralization by antibody. Three distinct sites have been identified which are designated site 1, site 2 and site 3. Site 1 includes a region of 12 amino acids of VP1, from residues 89 to 100, and a corresponding region of VP1 has been identified as an antigenic site for poliovirus 2. This site was strongly immunodominant in type 2 and type 3 but was not detected for poliovirus 1. Site 2 is a complex site including residues 220 to 222 from VP1 (site 2a) with residues including 169 and 170 and others of VP2 (site 2b). Both site 2a and site 2b have been detected in type 1 poliovirus, while as yet only site 2b has been detected in type 3 poliovirus. Site 3 is a complex site including residues 286 to 290 from VP1 (site 3a) with residues including 58 and 59 and others of VP3 (site 3b). Both sites 3a and 3b have been detected in type 3 poliovirus, while as yet only site 3b has been detected in type 1 poliovirus.

Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

ABSTRACT The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel polioviruscis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

Synthesis of Immunogenic, but Non-infectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector

A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses.

Monoclonal antibodies which block cellular receptors of poliovirus.

The isolation and properties of monoclonal antibodies which specifically inhibit the binding of poliovirus types 1, 2 and 3 to cells are described. The antibodies were of the IgG class and blocked infection of cells by all strains of the three poliovirus serotypes, but by none of a wide range of other viruses examined, including nine human enteroviruses. The antibodies prevented poliovirus growth in all susceptible human and primate cells tested. We conclude that the antibodies are directed against the receptor site for poliovirus which is distinct from those required by other picornaviruses, and which seems to be antigenically well conserved between cells of human and primate origin.

The Temperature Sensitivity of the Sabin Type 3 Vaccine Strain of Poliovirus: Molecular and Structural Effects of a Mutation in the Capsid Protein VP3

The growth of the Sabin strain of type 3 poliovirus is reduced at high temperatures compared to that of its virulent precursor strain Leon. Recombinant viruses have been generated from infectious cDNA clones and demonstrate that the temperature-sensitive (ts) phenotype is mainly attributable to a difference in residue 91 of the virion protein VP3. Examination of non-ts mutants derived in vitro or in vivo reveals the existence of second site mutations some of which are clearly able to suppress the ts phenotype. The location of residue 91 of VP3, and of a number of candidate suppressor mutations, in the atomic structure of the virion suggests that the ts phenotype may result in destabilization of the particle and that the suppressors may function by stabilizing specific interfaces. It is not yet clear whether the ts phenotype is expressed at the level of the particle or in the form of defects in assembly or uncoating of the virion, or all three.

Rational Design of Genetically Stable, Live-Attenuated Poliovirus Vaccines of All Three Serotypes: Relevance to Poliomyelitis Eradication

ABSTRACT The global eradication of poliomyelitis caused by wild-type virus is likely to be completed within the next few years, despite immense logistic and political difficulties, and may ultimately be followed by the cessation of vaccination. However, the existing live-attenuated vaccines have the potential to revert to virulence, causing occasional disease, and viruses can be shed by immunocompromised individuals for prolonged periods of time. Moreover, several outbreaks of poliomyelitis have been shown to be caused by viruses derived from the Sabin vaccine strains. The appearance of such strains depends on the prevailing circumstances but poses a severe obstacle to strategies for stopping vaccination. Vaccine strains that are incapable of reversion at a measurable rate would provide a possible solution. Here, we describe the constructions of strains of type 3 poliovirus that are stabilized by the introduction of four mutations in the 5′ noncoding region compared to the present vaccine. The strains are genetically and phenotypically stable under conditions where the present vaccine loses the attenuating mutation in the 5′ noncoding region completely. Type 1 and type 2 strains in which the entire 5′ noncoding regions of Sabin 1 and Sabin 2 were replaced exactly with that of one of the type 3 strains were also constructed. The genetic stability of 5′ noncoding regions of these viruses matched that of the type 3 strains, but significant phenotypic reversion occurred, illustrating the potential limitations of a rational approach to the genetic stabilization of live RNA virus vaccines.

Induction by synthetic peptides of broadly reactive, type-specific neutralizing antibody to poliovirus type 3.

A region of virus capsid protein VP1 located 89-100 amino acids from the N-terminus has been proposed to comprise a major antigenic site involved in the neutralization of poliovirus type 3. Synthetic peptides 10-18 amino acids in length, containing all or part of this sequence, were tested for their ability to induce antiviral antibodies. Rabbits, but not guinea pigs or mice, immunized with the most active peptide, developed hightitered, type-specific, neutralizing antibodies for a wide range of poliovirus type 3 strains. Consistent with the broad type specificity of the antibody response was the observation that amino acids 89-100 of VP1 are highly conserved among different poliovirus type 3 strains. This sequence thus appears to provide, at least in part, a molecular basis for serotype antigenic specificity. Individual amino acids from 93 to 98 within this sequence were shown to be important for the neutralization of virus by antipeptide sera by examination of the ability of the sera to neutralize laboratory-derived poliovirus type 3 mutants with known single amino acid substitutions in the proposed antigenic site.

Nucleotide sequence of the 16S ribosomal RNA gene from the haloalkaliphilic archaeon (Archaebacterium) Natronobacterium magadii, and the phylogeny of halobacteria

Summary The ribosomal RNA operon of the haloalkaliphilic archaeon Natronobacterium magadii was cloned into plasmid vectors. Southern analysis of the cloned genes revealed an organization similar to that of other halobacteria, with rRNA genes linked in the eubacterial order, with a putative tRNA ala located in the 16S–23S intergenic space. The primary sequence of the 16S rRNA which was determined by DNA sequencing, can be folded to give a secondary structure consistant with the universal structure proposed by Woese et al. (1983). The 16S rRNA gene is flanked by three sets of inverted repeats which may be involved in 16S rRNA maturation. The nucleotide sequence of the 16S rRNA gene together with previously published sequences was used to produce a phylogenetic tree for the halophilic archaea (halobacteria).

Cloning of the genome of cricket paralysis virus: sequence of the 3′ end

Abstract DNA complementary to the single-stranded RNA genome of the insect picornavirus, cricket paralysis virus (CrPV), was cloned into the plasmid pBR322 by a hybrid RNA-cDNA cloning strategy. Positive CrPV-specific clones were selected by colony hybridization and characterised by restriction enzyme mapping. Overlapping clones spanning 7.5 kb of the estimated 8.5 kb genome were obtained, the largest being 7.0 kb. Comparison of the restriction enzyme map with those of mammalian picornaviruses revealed no conserved pattern of cleavage sites. The CrPV-cDNA was sequenced using the M13-dideoxy chain-terminating method although the chemical method of Maxam and Gilbert was employed to complete gaps in the sequence. The sequence of the 3′-terminal 1600 nucleotides is presented and is compared with those of mammalian picornaviruses. Computer comparisons of the CrPV sequence and those of mammalian picornaviruses revealed no significant homology between either the nucleotide or the predicted amino acid sequence of this region.

The Structural and Infected Cell Polypeptides of Influenza B Virus

Structural and virus-induced infected cell polypeptides of several strains of influenza B virus were examined by high resolution polyacrylamide gel electrophoresis and shown to be directly analogous to those of the influenza A viruses. Eight structural polypeptides, P1, P2, P3, HA1, HA2, NA, NP and M were observed in purified virus and at least two additional polypeptides, HA and NS could be detected in infected MDCK cells. The three P proteins plus NP were shown to be associated with RNA-dependent RNA polymerase activity and HA, HA1, HA2 and NA were shown to be glycosylated. Like the influenza A viruses, migrational differences of some of the infected cell polypeptides could be observed between different B strains. Investigation of a time course of virus replication failed to show any temporal control of protein synthesis in the infected cell.