Systematic nomenclature of picornavirus proteins.

An easily learned convention for systematizing the nomenclature of picornavirus proteins is described. The convention is based upon an idealized map, called the L434 diagram, of the picornavirus polyprotein.

Single major polypeptide of a calicivirus: characterization by polyacrylamide gel electrophoresis and stabilization of virions by cross-linking with dimethyl suberimidate.

A calicivirus, San Miguel sea lion virus serotype 4, isolate 15FT, externally labelled with 125I, was shown by gel electrophoresis to possess a single major polypeptide. The polypeptide migrated anomalously upon electrophoresis in two sodium dodecyl sulfate (SDS) systems: more slowly than bovine serum albumin in a continuous phosphate-buffered system and more rapidly than bovine serum albumin in a discontinuous system. Estimated molecular weights in the two systems were approximately 71,000 and 64,000, respectively. There was no clear evidence for a minor virion polypeptide. Treatment of purified San Miguel sea lion virions with dimethyl suberimidate, a cross-linking reagent, preserved virion integrity during long-term storage at 4 degrees C. Oligomeric species of the polypeptide were observed upon electrophoresis of products from cross-linked virions. Based upon a preferred polypeptide molecular weight estimate of 71,000 and distribution of oligomeric species, a calicivirion model with 120 monomeric protein units is proposed as an alternative to a 180-unit model.

Alignment of protein sequences using secondary structure: a modified dynamic programming method.

A method for comparison of protein sequences based on their primary and secondary structure is described. Protein sequences are annotated with predicted secondary structures (using a modified Chou and Fasman method). Two lettered code sequences are generated (Xx, where X is the amino acid and x is its annotated secondary structure). Sequences are compared with a dynamic programming method (STRALIGN) that includes a similarity matrix for both the amino acids and secondary structures. The similarity value for each paired two-lettered code is a linear combination of similarity values for the paired amino acids and their annotated secondary structures. The method has been applied to eight globin proteins (28 pairs) for which the X-ray structure is known. For protein pairs with high primary sequence similarity (greater than 45%), STRALIGN alignment is identical to that obtained by a dynamic programming method using only primary sequence information. However, alignment of protein pairs with lower primary sequence similarity improves significantly with the addition of secondary structure annotation. Alignment of the pair with the least primary sequence similarity of 16% was improved from 0 to 37% 'correct' alignment using this method. In addition, STRALIGN was successfully applied to seven pairs of distantly related cytochrome c proteins, and three pairs of distantly related picornavirus proteins.

Absence of poly (A) from the infective RNA of Nodamura virus.

With the exception of phage Qbeta, the RNAs of all the other small icosahedral RNA viruses so far examined contain a poly (A) tract. This tract has been implicated in the infectivity of poliovirus RNA. We have now shown that Nodamura virus, a divided genome virus from which infective RNA can be extracted, does not contain any poly (A) tracts. This evidence with Nodamura virus shows that poly (A) is not a necessary requirement for the infectivity of virus RNA molecules.

A major difference in the strategy of the calici- and picornaviruses and its significance in classification.

Pig kidney (1BRS-2) cells infected with vesicular exanthema virus (VEV), a calicivirus, did not contain any large precursor polypeptides similar to those found when they were infected with foot-and-mouth disease virus (FMDV). The largest induced protein found in the VEV-infected cells had a molecular weight identical with that of the virus structural polypeptide. This difference in strategy between VEV and FMDV, taken in conjunction with the morphological and structural differences described previously, provides strong evidence that the caliciviruses should not be included in the family Picornaviridae.

RNA synthesized in calicivirus-infected cells is atypical of picornaviruses.

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.

Detection of picornavirus sequences in nervous tissue of amyotrophic lateral sclerosis and control patients.

We used in situ hybridization to look for picornavirus ribonucleic acid (RNA) sequences in frozen sections of central nervous system (CNS) tissue of amyotrophic lateral sclerosis (ALS) and control patients. Using reconstruction experiments, we concluded that 30 copies of viral RNA per cell could be detected with the assay. RNA which hybridized to DNAs complementary (cDNAs) to both poliovirus and Theiler's virus was found at several levels in the CNS of 2 patients, 1 ALS patient, and 1 control. In transverse sections of the spinal cord, these sequences predominated in cells of the anterior horns. We assessed the specificity of hybridization by several criteria: no hybridization was observed with heterologous visna virus cDNA probes; hybridization was abolished by pretreatment of the sections with ribonuclease; chemography artifacts were ruled out; and the results were reproduced in three independent experiments. We concluded that RNA molecules, possibly belonging to a picornavirus having sequences in common with poliovirus and Theiler's virus, were present in the CNS of these 2 patients. On the other hand, 14 cases of classic ALS, 2 cases of Guamanian parkinsonian dementia, and 5 controls had negative results. However, the presence of picornavirus sequences in our series could be underestimated because in many cases autolysis times were 10 hours or longer.

Relationship of San Miguel sea lion virus to other members of the calicivirus group.

San Miguel sea lion virus (SMSV) is indistinguishable from vesicular exanthema virus (VEV) and feline calicivirus (FCV) in its morphology and in possessing a single capsid polypeptide with a molecular weight of approximately 65 X 10(3). Neutralization tests readily differentiate the three viruses, but immunodiffusion tests show that SMSV is closely related serologically to VEV but not to FCV. Although the RNAs of the three caliciviruses have similar base compositions, homology tests show that SMSV is closely related to VEV but is not related to FCV. Tryptic peptide maps of the single major polypeptide comprising the capsid of each virus also show that SMSV and VEV are more closely related to each other than to FCV.

In vitro translation of the Two RNAs of Nodamura virus, a novel mammalian virus with a divided genome.

Nodamura virus is a small ribovirus containing two RNA molecules. Both RNAs were found to be active messengers for protein synthesis in cell-free extracts prepared from wheat embryo or HeLa cells. RNA 2 directed synthesis of a 43,000-dalton product, p43, whose tryptic fingerprint was similar to that of the major viral coat protein, vp40 (molecular weight, 40,000). Though p43 appears to be a precursor of vp40, processing did not occur in the cell-free extracts. RNA 1 directed synthesis of a 105,000-dalton protein, p105. Its tryptic fingerprint revealed no evidence of coat protein sequences. Hence, the two RNAs represent genes with few, if any, redundant coding sequences.

Further physicochemical characterization of Nodamura virus. Evidence that the divided genome occurs in a single component.

Nodamura virus, a small non-enveloped RNA virus, contains two species of RNA sedimenting at 22S (RNA-1) and 15S (RNA-2), a single major polypeptide of mol. wt. 40 X 10(3) and two minor polypeptides, of mol. wt. 38 and 43 X 10(3). Evidence is presented that the two RNA species are in the same particle. Although extraction of the virus with SDS-phenol yields the two species of RNA as separate entities, gentle treatment of the virus with guanidine and low concentrations of SDS releases the RNA as a 27S component which contains both RNA-1 and RNA-2 together with a trace of protein. It seems likely that the two RNA species replicate separately because double stranded molecules corresponding to the single stranded RNA-1 and RNA-2 molecules were present in BHK cells infected with the virus.

Physicochemical characterization of two serologically unrelated equine rhinoviruses.

The physicochemical properties of two serologically distinct equine rhinoviruses have been examined. Each virus sedimented at approximately 160S but co-centrifugation of the two viruses in a sucrose gradient revealed a small difference in their sedimentation coefficients. The two viruses also have different buoyant densities in cesium chloride. The equine rhinovirus type 1 equilibrated as a sharp peak at 1.45 g/ml whereas the type 2 virus equilibrated as a heterogeneous band with a peak at 1.44 g/ml but ranging in density from 1.41 to 1.45 g/ml. The relative sedimentation coefficients of the two virus RNAs were 35S for rhinovirus 1 and 37S for rhinovirus 2. A limited number of base composition analyses also showed differences between the two virus RNAs. The polypeptide profile of each serotype in polyacrylamide gels was generally similar to those of other picornaviruses but the two serotypes could be distinguished readily from each other.