- Volume 72, Issue 4, 1991

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neuro-virulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.

Proteolytic activation of Sendai virus in the lungs of mice is necessary to cause pneumopathogenicity. Using Sendai virus-infected lung block cultures, protease inhibitors were tested for their antiviral effect by examining inhibition of proteolytic activation. Among the inhibitors tested, a serine protease, aprotinin, was shown to be most effective. In vivo protection experiments demonstrated that aprotinin, when administered intranasally, could confer protection on mice against lethal Sendai virus pneumonia through the same mechanism as observed in the in vitro system. The present study provides an experimental basis for the use of protease inhibitors as antiviral drugs.

The complete nucleotide sequence of the nucleoprotein (NP) gene of human parainfluenza virus type 1 (hPIV-1) was determined from a cDNA clone of mRNA. The mRNA is 1683 nucleotides long (excluding polyadenylic acid) and encodes a protein of 524 amino acids with a predicted M r of 57548. An amino acid identity of 83% was predicted between the NPs of the human pathogen hPIV-1 and the murine paramyxovirus, Sendai virus, compared to 72% similarity at the level of the nucleotide sequence. In contrast, the amino acid sequence identity between the NPs of hPIV-1 and hPIV-3 was 59%, suggesting a more distant evolutionary relationship. The NP amino acid sequences of hPIV-1 and Sendai virus were highly conserved in the amino-terminal half of the molecule, in which 395 of the first 420 amino acids were identical. Of 11 monoclonal antibodies (MAbs) targeted against the Sendai virus NP, five cross-reacted with the hPIV-1 NP. The MAbs that cross-reacted recognize epitopes within regions of high amino acid similarity between the NPs of the two viruses. Also, five of the eight MAbs raised against hPIV-1 NP cross-reacted with Sendai virus NP. Taken together, our observations suggest that the essential amino acid sequence determinants of the NP structures of hPIV-1 and Sendai virus are conserved despite changes in their nucleotide sequences during evolution. This implies that there was a selective pressure to maintain the important functional domains of the protein.

The RNA 3 of rice stripe virus (RSV) isolate T was cloned and its nucleotide sequence was determined. The complete primary structure was found to consist of 2504 nucleotides. One putative open reading frame (ORF), between nucleotides 2412 and 1444 in the 5′-proximal region of the virus complementary-sense RNA, encoded a 322 amino acid protein with an M r of 35134 that was identified as the coat protein. The other ORF, between nucleotides 66 and 701 in the 5′-proximal region of the virus-sense RNA, encoded a 211 amino acid protein with an M r of 23 874. An intergenic non-coding region (742 nucleotides) between the two ORFs contains oligo(A)- and oligo(U)-rich regions that can be arranged into a 126 base pair stem configuration. These results suggest that RSV RNA 3 has an ambisense coding strategy.

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5′ nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5′ oligonucleotide marker (Ω) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions ‘destabilized’ by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5′ leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that Ω at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3′ to, the first AUG codon. We propose that the complete 5′ leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.

RNA 2 of the V strain of tomato aspermy virus (TAV) consists of 3074 nucleotides and contains one open reading frame of 2487 nucleotides. Thus, it resembles RNA 2 of cucumber mosaic virus (CMV) strains Q and Fny (62% identical to both), brome mosaic virus (42% identical) and cowpea chlorotic mottle virus (40% identical). In comparisons between amino acid sequences, three different regions of similarity could be distinguished. These were the central part (amino acids 224 to 757 for V-TAV), which was most similar among the four viruses, and the N and C ends; sequences conserved among RNA polymerase species were found in the C half of the central part. Hydrophobicity patterns, and distributions of acidic and basic amino acids in the proteins encoded by V-TAV RNA 2, Q-CMV RNA 2 and Fny-CMV RNA 2 were very similar except at the extreme ends of the molecules. Structures that have been reported to act as regulatory signals for minus- and plus-strand synthesis were found in the 5′ and 3′ non-coding regions of the RNA.

In vitro synthesis of plum pox potyvirus (PPV)-specific nucleic acid has been measured in a crude fraction prepared from leaves of PPV-infected Nicotiana clevelandii plants. Using alkali and DNase treatments, the synthesized nucleic acid was shown to be RNA. The electrophoretic mobility and the differing sensitivity to RNase at high and low salt concentrations allowed the identification of in vitro products probably corresponding to replicative form and replicative intermediate RNA, as well as to single-stranded RNA. Most of the PPV-specific RNA synthesized was shown to be of positive polarity. The in vitro RNA synthesis, performed in the presence of actinomycin D, required all four ribonucleoside triphosphates and Mg2+ ions. This enzyme extract contained about 6% of the leaf protein and most of the identified virus-encoded proteins. Altogether, the results presented in this paper suggest that in vitro RNA synthesis was carried out by the PPV replicase complex.

ELISA, cDNA dot blot hybridization and transmission by vector aphids were used to investigate the occurrence and degree of cross-protection produced in oat plants by virus isolates representing five strains or serotypes of barley yellow dwarf virus, namely PAV, MAV, SGV, RPV and RMV. Generally, the degree of cross-protection was positively correlated with the serological relatedness between the isolates. A high degree of cross-protection occurred between NY-MAV and MAV-PS1, two isolates of the MAV serotype; cross-protection was moderate between MAV-PS1 and either P-PAV (a Purdue isolate of the PAV serotype) or NY-SGV; cross-protection between P-PAV and NY-SGV was low. Cross-protection did not occur in other paired inoculations and did not persist in some plants, the challenge virus eventually becoming detectable. The persistence of cross-protection depended on the interval between inoculations with protecting and challenge viruses; longer inoculation intervals enhanced the persistence of cross-protection. Results obtained by ELISA and dot blot hybridization were usually consistent, indicating that cross-protection affected both viral capsid and RNA synthesis.

The complete nucleotide sequence of RNA 2 of a German isolate of barley yellow mosaic virus (BaYMV) has been determined. The RNA is 3585 nucleotides in length excluding a 3′-terminal poly(A) tail. The viral plus and minus strands in all three reading frames contained only one large open reading frame which started at positions 156 to 158 and terminated with a UAG codon at positions 2828 to 2830, thus encoding an M r 98000 polypeptide. Comparisons with sequences of other viruses revealed that the amino terminus of the polypeptide has homology with the proteolytic domain of the helper component proteinase of several potyviruses. It was determined that the 98K protein is a polyprotein composed of an amino-terminal 28K protein and a 70K protein.

The sequence of the 3585 nucleotides [excluding the 3′ poly(A) tail] of barley yellow mosaic virus (BaYMV) RNA 2 was obtained by analysis of cDNA clones and by direct RNA sequencing. The first initiation codon at nucleotide 155 was followed by a single long open reading frame encoding a protein of 890 amino acids with an M r of 98458. Amino acid sequence comparisons indicated that the BaYMV 98K protein contains a region similar to the C-terminal proteinase domain of the potyvirus helper component (HC) protein towards its N terminus, but that it has no sequences that resemble the N-terminal part of the HC protein or other proteins of potyviruses. The data reveal striking differences in genetic organization between BaYMV RNA 2 and the 5′-terminal region of the potyvirus genome, despite a close genetic relationship between BaYMV RNA 1 and the rest of the potyvirus genome.

The nucleotide sequences of the coat protein genes and 3′ non-coding regions of two aphid-transmissible isolates (G2 and G7) of soybean mosaic virus (SMV) were determined. The coat protein of the G2 isolate differs from that of the aphid non-transmissible N isolate by a single amino acid at position 12 (aspartic acid in N, glycine in G2 and G7). The G7 isolate differs from G2 and N at three and four amino acid residues, respectively. The nucleotide sequence similarity of the three isolates in the coat protein coding and 3′ non-coding regions ranges from 93 to 100%, respectively. In contrast, watermelon mosaic virus 2 (WMV 2) is only 77% to 79% similar to the SMV isolates, suggesting that WMV 2 is a distinct virus and not an isolate of SMV as proposed previously.