- Volume 71, Issue 11, 1990

Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid. Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants. Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and LI (three regions) open reading frames could be found by this approach. Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids. In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum. Using an approach to predict ‘receptor-like’ regions within the respective proteins, five of the seven regions were also identified. From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA.

Nine overlapping peptides (20 amino acid) covering the entire sequence of early antigen E7 of human papillomavirus type 16 (HPV-16) were synthesized and tested as antigens with human sera in ELISA. Five of these peptides (no. 1 to 5 counting from the N terminus of the E7 protein) reacted with a pool of sera from HPV-16-infected individuals (as determined by molecular hybridization with their biopsy specimens); one (no. 5) was also reactive with pools of HPV-18- and HPV-6- or 11-infected individuals. Sera from 24 patients with cervical intraepithelial neoplasia (CIN) and from 29 invasive cervical carcinoma (INCA) patients were tested for the presence of antibodies reactive with peptides, no. 1 to 4 covering amino acids 1 to 50 and with peptide no. 5 covering amino acids 41 to 60. Only one of the sera from CIN patients was reactive with peptides no. 1 (amino acids 1 to 20) and no. 4 (amino acids 31 to 50). However, the majority of these sera reacted with peptide no. 5. The occurrence of this antibody was only slightly less frequent in sera from healthy subjects compared to CIN patients. On the other hand, sera from the INCA patients were reactive with the peptides no. 1 to 3 more frequently than the sera from matched control subjects. Positive reactions of sera from INCA patients were most frequently seen with no. 2; 24% of these sera but only 7% of the controls were reactive with no. 2 peptide. The present data suggest that no. 1 to 3 are HPV-16-specific, whereas no. 5 is broadly cross-reactive.

The L2 open reading frames (ORFs) of human papillomavirus (HPV) types 6b and 11 were expressed as full-length non-fusion proteins in Spodoptera frugi- perda (Sf-9) cells using recombinant baculovirus. Both proteins were detected on Western blots as immuno- reactive bands which migrated with apparent Mr s of 76K and 78K, respectively, and contained both crossreactive and type-specific epitopes, as determined by polyclonal antisera directed against defined subregions of the HPV-6b and HPV-11 L2 ORFs. In addition, the minor capsid protein of HPV-11 particles co-migrates with the HPV-11 L2 ORF product and is immunore- active with HPV-11 L2-specific antisera. These observations indicate that the anomalous electrophoretic mobilities of papillomavirus L2 ORF proteins can be explained without invoking post-transcriptional processing events and that the minor capsid protein of HPV-11 is antigenically and biophysically related to the HPV-11 L2 ORF product.

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi’s sarcoma and in cell lines from brain tumours, Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable element in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

The topographical distribution of the poliovirus receptor on the cell surface was demonstrated by immunoelectron microscopy using monoclonal antibodies and immunogold markers. The receptor appeared in small clusters, which were randomly distributed over the cell surface and along cellular processes. The distribution pattern of the clusters corresponded to that of adsorbed and immunogold- labelled poliovirus particles and suggests a multivalent organization of poliovirus binding sites. Freezefracturing and ultrathin sectioning did not reveal any specific ultrastructures within the plasma membrane at labelled receptor areas. Incubation of native cells with anti-receptor antibodies did not remove the receptor molecule from the cell surface nor did it induce ultrastructural alterations within the plasma membrane. The antibody-receptor complexes exhibited lateral mobility within the plasma membrane and were able to aggregate into large immune complexes after incubation with a second ligand.

Synthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP′2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific antibodies could be induced in BALB/c mice with a synthetic peptide representing the epitope. The potential of this non-dominant B cell epitope to induce antiviral immunity in the presence of maternal CPV-specific antibodies, is discussed.

The nucleotide sequence of feline panleukopenia virus (FPV) strain 193 was determined and compared with the sequence of canine parvovirus (CPV) strain N and partial sequences of FPV strain Carl and CPV strain b. Base differences were identified at 115 positions in these 5·1 kb genomes and predicted amino acid differences occurred at 40 positions. The two overlapping capsid protein genes contained almost twice as many base differences as the single non-structural protein gene (49 compared to 26) and about the same ratio was calculated for predicted amino acid differences (27 compared to 13). The 27 variant amino acids in the capsid proteins were clustered at three sites in the primary sequence, whereas 10 of the 13 variant amino acids in the non-structural protein occurred in the 130 C-terminal amino acids. The two FPV strains differed consistently from the two CPV strains at 31 bases: 12 base changes in the capsid protein genes resulted in six amino acid changes, six base changes in the nonstructural protein gene resulted in three amino acid changes, and 13 base changes occurred in the noncoding sequence.

The hepatitis B virus particle consists of an envelope carrying the surface antigen of the virus and an internal capsid consisting of the core antigen (HBcAg). The internal capsid contains the circular, partially dsDNA genome and the viral polymerase. Empty core particles have been produced in Spodoptera frugiperda cells using a recombinant baculovirus vector, YMIKTc, that expresses a 21· 4K derivative of the HBcAg gene. The particles have been purified to homogeneity by caesium chloride density gradient centrifugation followed by glycerol gradient centrifugation. Physicochemical analysis of the core particles showed that they exhibited a sedimentation coefficient (s0 20,w) of 82·5S and a diffusion coefficient (D) of 1 ·28 × 10−7 cm2/s. The M r obtained by substitution of these values in the Svedberg equation was 5·8 × 106, using a partial specific volume of 0·73 ml/g for the viral protein as estimated from the amino acid composition. The M r determined from sedimentation equilibrium analyses was 6·3 × 106. Spectrophotometric and metabolic labelling analyses failed to detect nucleic acids in the core preparations. The data are at variance with the prediction that cores exhibit a T=3 symmetry and contain some 180 subunits. The results suggest that the baculovirus- expressed cores may contain up to 300 subunits of HBcAg protein.

A murine model based on infection by the respiratory route has been used to study the pathogenesis of recombinant vaccinia viruses. The neurovirulent Western Reserve (WR) strain and the Wyeth smallpox vaccine strain were used as vectors. Recombinant viruses were constructed by insertion of the Epstein- Barr virus membrane glycoprotein 340 gene into the thymidine kinase (TK) gene of each vaccinia virus. Intranasal inoculation of DBA/2 mice with 106 pockforming units (pk.f.u.) of the WR strain was lethal but mice survived similar infection with the WR recombinant virus. Each virus was recovered from lung, blood and brain but, unlike wild-type virus, the recombinant virus was subsequently cleared. No deaths occurred after similar infection with the Wyeth strain or the Wyeth recombinant virus. There was limited growth of the Wyeth strain in the respiratory tract, low levels of virus in the blood and only sporadic recovery in brain extracts. The Wyeth recombinant virus was cleared rapidly with little viraemia or detectable infection of the central nervous system. No phenotypic character determined in vitro could be related consistently to the virulence of wild-type and recombinant viruses. Although the lethal character of the WR strain was affected by its TK+ phenotype, mice survived infection by intranasal inoculation with 106 pk.f.u. of WR TK+ recombinant viruses which either expressed the human interleukin 2 gene or had a deficient vaccinia virus growth factor gene.

The baculovirus expression system has been used to produce non-structural proteins encoded by bottom- component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (/lcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.

The application of the polymerase chain reaction DNA amplification technique to the detection and typing of isolates of maize streak virus (MSV) and other related geminiviruses of grasses is described. The oligonucleotide primers used for amplification were 17-mers which contained a number of degeneracies. An approximately 250 base pair fragment was amplified from all geminivirus-infected grass and cereal samples tested. The amplification reaction was specific, working down to a concentration of 50 fg/ml of MSV-specific plasmid-cloned DNA and with a 10−9 dilution of MSV- infected maize DNA extract. DNA could also be amplified from distantly related geminiviruses, including two different sugarcane viruses, digitaria streak virus and another as yet uncharacterized virus of a Panicum sp. Amplified DNA from a Mauritian sugarcane isolate (SSV-M) was cloned and sequenced. Sequence comparison and phylogenetic analysis showed that this sequence differed sufficiently from the analogous region of other geminiviruses for SSV-M to be considered a distinct virus. The use of the polymerase chain reaction for the amplification of gemini- and other virus genomes or genomic fragments for typing, mapping, phylogenetic analysis and taxonomy is discussed.

DNA complementary to the 3′-terminal 3684 nucleotides of the Ornithogalum mosaic potyvirus (OMV) genome was cloned and sequenced. The sequence consisted of a single large open reading frame which probably starts upstream of the cloned region. By comparison to other sequenced potyviruses, it was estimated that the clone contained the 3′ non-coding (3′-NC) region, the coat protein (CP) gene and the large nuclear inclusion protein (NIb) gene, as well as approximately 85% of the small nuclear inclusion protein (NIa) gene. The 3′-NC region of 274 nucleotides showed 38% to 45% similarity to the corresponding regions of other potyviruses. The putative CP gene could encode a 253 amino acid coat protein with a calculated M r of 28807. Analysis of the amino acid sequences of OMV and other potyvirus proteins showed similarities of 66% to 77% for CP, 72% to 73% for NIb and 63% to 71% for NIa proteins. These data, as well as phylogenetic analysis of the CP sequences, suggested that OMV is a typical but taxonomically distinct potyvirus.

A sequence of 1801 nucleotides originating from the 3′ end region of turnip mosaic virus (TuMV) RNA was cloned using the polymerase chain reaction and found to contain one long open reading frame (ORF). The amino acid sequence of three different regions of the isolated TuMV capsid protein (including the NH2 terminus) was determined and these partial sequences were found in the translation product predicted to be encoded by the large ORF. The data suggested that the TuMV capsid protein was a product arising from the maturation of a larger polyprotein, as observed for other potyviruses. Furthermore, the putative cleavage site corresponded to a glutamine-alanine dipeptide, a site commonly used in plant virus polyprotein processing. The capsid protein cistron was composed of 864 nucleotides and corresponded to a region encoding 288 amino acids with a calculated M r of 33186; the adjacent 3′ non-coding region was 667 nucleotides long. The deduced amino acid sequence of the TuMV capsid protein is closely related to other potyvirus capsid proteins, with most of the variation being found within the NH2-terminal region.