Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.

Poliovirus vectors are being studied as potential vaccine delivery systems, with foreign genetic sequences incorporated as part of the viral genome. The foreign sequences are expressed as part of the viral polyprotein. Addition of proteolytic cleavage sites at the junction of the foreign polypeptide and the viral proteins results in cleavage during polyprotein processing. The ability of foot-and-mouth disease virus (FMDV) 2A to mediate proteolytic cleavage in the context of poliovirus vectors was studied. The results demonstrate that FMDV 2A is able to generate cleavage of the foreign antigen from the viral polyprotein. A second cleavage event between the FMDV 2A peptide and the foreign protein was also observed.

Characterization of recombinant polioviruses expressing regions of rotavirus VP4, hepatitis B surface antigen, and herpes simplex virus type 2 glycoprotein D.

Recombinant polioviruses expressing antigens from rotavirus, herpes simplex virus type 2, and hepatitis B virus were generated. Fusion of the heterologous polypeptides to the amino terminus of the poliovirus polyprotein did not prevent myristylation of VP0, suggesting a novel mechanism of myristylation for these recombinant viruses. The effects of the parental genetic background, different foreign sequences, and different insert sizes on growth characteristics were compared. Both the size and the nature of the heterologous sequence appeared to be factors influencing the growth and stability of recombinant polioviruses. All of the recombinants showed a temperature-sensitive phenotype, regardless of the genetic background (attenuated or wild type) from which they were derived. Preliminary studies with transgenic mice carrying the poliovirus receptor gene are discussed.

Attenuated poliovirus strain as a live vector: expression of regions of rotavirus outer capsid protein VP7 by using recombinant Sabin 3 viruses.

The ability to express heterologous antigens from attenuated poliovirus strains suggests the potential for use as live vectored vaccines. Full- or partial-length sequences of the gene encoding rotavirus major outer capsid protein VP7 were cloned into the open reading frame of a full-length cDNA copy of poliovirus Sabin type 3. They were inserted either at the 5' end or immediately after the capsid protein coding region, at the junction between precursors P1 and P2. A protease cleavage site for 3C protease was introduced 3' to the foreign sequences to enable proteolytic processing of the antigen from the poliovirus polyprotein. Infectious viruses were generated from several of the DNA constructs, and the presence of the foreign gene sequences was confirmed by reverse transcription of the viral RNA and PCR amplification. Viruses with inserts of about 300 bases maintained the foreign sequences during passage in Vero cells. Viruses carrying larger sequences were unstable, and deletions were generated within the foreign sequences. Expression of the VP7 polypeptides was demonstrated by immunoprecipitation with specific antiserum of labeled proteins from cells infected with Sabin 3 recombinant viruses. Comparative studies of RNA synthesis showed similar kinetics for Sabin 3 and the Sabin 3/VP7 recombinants. One-step growth curves showed that production of recombinant viruses was slower than that of Sabin 3 and that the final titers were 1 to 1.5 logs lower. Accumulation of VP7-containing precursors in infected cells suggests that slow cleavage at the engineered 3C protease site may be a limiting step in the growth of these recombinant Sabin polioviruses and may influence the permissible size of foreign sequence to be inserted.

A subviral particle binding domain on the rotavirus nonstructural glycoprotein NS28.

The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.

Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.

Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.

Alterations in the sequence of the gene 4 from a human rotavirus after multiple passages in HepG2 liver cells.

The human Wa strain of rotaviruses, initially unable to grow in liver cells, was adapted by multiple passages to grow in HepG2 cells. The genome segment 4 of both the parental and passaged strains was cloned and sequenced. Five amino acid differences (residues 38, 120, 421, 525, and 618) were found in the HepG2-passaged variant compared to the parental Wa strain. Our results support the hypothesis that viral variants that have improved capabilities for infecting liver cells can be generated during infection.

Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34.

Intermolecular interactions between polypeptide chains often play essential roles in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly. We have initiated studies of the functions of the rotavirus SA114F gene 7 product by sequence analysis and expression in insect cells. This nonstructural protein, NS34, is a slightly acidic protein, and its secondary structure is predicted to be 78% alpha-helix, with several heptad repeats of hydrophobic amino acids being present in its carboxy half. NS34 was found in oligomers when analyzed in insect cells, in SA11-infected MA104 cells, and in cell-free translation reactions. Investigation of the multiple electrophoretically distinct forms of NS34 showed they were all composed of homooligomers. Deletion mutants constructed and tested for oligomerization showed that the carboxy terminus of the protein, containing the predicted heptad repeats, was responsible for oligomerization. A basic region present in NS34 of group A rotaviruses, found to be 40% conserved in NS34 of group C rotavirus, is a candidate for a functional domain of this protein. NS34, which was found to be associated with the cytoskeleton fraction of cells, also interacts with viral RNA. These results make it likely that NS34 plays a central role in the replication and assembly of genomic RNA structures.

Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity.

We have examined the possible function(s) of the protein VP3 encoded by the rotavirus SA11 genomic segment 3. Viral-associated VP3 in double-shelled and single-shelled particles was shown to bind GTP covalently and reversibly. These properties are similar to the unique characteristics of eukaryotic and viral guanylyltransferases, suggesting that VP3 is associated with a capping enzyme activity. Previous studies have shown that intact viral particles are required for transcription, making it difficult to unequivocally identify the functions of individual proteins within such particles. Characterization of VP3 produced in the baculovirus expression system showed that the expressed VP3 covalently bound GTP. These studies suggest that VP3 alone is the guanylyltransferase. GTP binding also was seen in core virus-like particles and single-shelled virus-like particles that lacked viral nucleic acid and were assembled in insect cells.

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Sequence of a rotavirus gene 4 associated with unique biologic properties.

The genome segment 4 of the simian rotavirus variant SA 11-4F was sequenced. This gene is of probable bovine origin, and it contains a few amino acid differences when compared with other SA 11 variants (4 fm and fem) that were isolated independently and that have fast migration patterns of their gene 4 segments. Hypotheses for the role of sequence changes are made relative to the unique properties of the SA 11-4F variant.

Porcine rotaviruses antigenically related to human rotavirus serotypes 1 and 2.

Fecal samples from rotavirus-infected piglets were characterized by a serotyping enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (MAbs) specific to human serotypes 1, 2, 3, and 4 (D. O. Matson, M. K. Estes, J. W. Burns, H. B. Greenberg, K. Taniguchi, and S. Urasawa, submitted for publication). Rotavirus in 19 of 25 specimens tested from two herds of pigs from Buenos Aires province, Argentina, were classified antigenically as follows: one serotype 1, four serotype 2, two serotype 3, and no serotype 4. Six specimens reacted with both serotype 1 and 2 MAbs, and viruses in six specimens probably belonged to other serotypes because they reacted only with a VP7 common epitope MAb. Two porcine rotavirus fecal samples found to contain both serotype 1 and 2 viruses by the MAb-based test and one found to contain a serotype 2 virus were grown in tissue culture. When plaque-purified preparations of these tissue culture-adapted viruses were analyzed in the serotyping ELISA, the C60 and C86 preparations reacted only as serotype 1 viruses, indicating that the original fecal samples, which showed multiple VP7 reactivities, were heterogeneous and apparently contained two types of viruses. Testing of plaque-purified C134 virus confirmed its serotype 2 reactivity. The MAb-based serotype designations of these viruses also were confirmed by using a neutralization immunoperoxidase focus reduction assay. This is the first report of the occurrence of serotype 1 and 2 rotaviruses in animals. The MAbs originally developed to serotype human rotaviruses can be utilized to type animal rotaviruses.

Genome rearrangements in porcine rotaviruses: biochemical and biological comparisons between a supershort strain and its standard counterpart.

Two porcine rotavirus strains (CN86 and CC86) isolated during an epidemiological survey of diarrhoea in swine in Argentina were studied because of several unique characteristics. Both these strains were isolated and cloned from the same faecal sample and the electrophoretic migration of 10 of their 11 genomic dsRNA genomic segments in polyacrylamide gels was identical, but strain CC86 had a supershort electropherotype. We analysed biochemical, serological and biological properties of both viruses. In vitro translation of genome segment 11 RNAs showed that both viruses produced a polypeptide with an apparent Mr of 26K. No differences in any of the other virus-induced proteins made in infected MA104 cells were found on one- and two-dimensional gels for either strain. In addition, the serotype and the subgroup specificities of both viruses were identical (group A, subgroup I, serotype 5). These results suggest that the rearranged strain was probably generated from the standard one and that the coding capacity of the rearranged segment was conserved. Consistent with this hypothesis, primer extension analysis revealed that the supershort strain had a rearrangement involving partial duplication of genomic segment 11. Biological studies showed differences between these viruses. The rearranged strain (CC86) produced larger plaques in monolayers of MA104 cells and outgrew the standard strain (CN86) when cells were coinfected with both viruses at different relative concentrations and different m.o.i. The possibility that large plaque formation and efficient virus replication can be influenced by the products of genomic segment 11, in addition to segment 4, is discussed.

Serological characterization of bovine rotaviruses isolated from dairy and beef herds in Argentina.

Bovine rotaviruses isolated from beef and dairy herds in Argentina were serotyped by the immunoperoxidase focus reduction assay as previously described (G. Gerna, M. Battaglia, G. Milenesi, N. Passarani, E. Percivalle, and E. Cattaneo, Infect. Immun. 43:722-729, 1984). Three strains from beef herds were related to the UK and NCDV bovine rotavirus strains defined as serotype 6 (Y. Hoshino, R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian, J. Infect. Dis. 149:694-702, 1984). Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983) in the United States. A serotyping antibody-capture enzyme-linked immunoassay to detect serotype 6 rotavirus using a serotype 6-specific monoclonal antibody was developed and evaluated for strain characterization. Characterization of 72 group A rotavirus-positive fecal samples from beef herds and 43 fecal samples from dairy herds showed a predominance of serotype 6 rotavirus in beef herds but both serotype 6 and non-serotype 6 rotaviruses in dairy herds. Analysis of genomic double-stranded RNA by polyacrylamide gel electrophoresis showed that when outbreaks were caused by one serotype only a single electropherotype was present in all samples.

Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism.

The structures of the rearranged genomic segment 11 of two spontaneous swine rotavirus strains were determined. We found that the rearrangements involved the duplication of normal segment 11 in a head-to-tail orientation, and partial deletions in both monomers. The open reading frame for VP11, the protein encoded by normal segment 11, was maintained. We also showed that the two rearranged genes were transcribed into RNA molecules of the same length as their corresponding genomic segments.

Antigenic characterization of swine rotaviruses in Argentina.

Fecal samples from 156 diarrheic piglets were collected from several herds located in two main breeding areas of Argentina. Rotaviruses were detected in 60 samples (38.4%) by polyacrylamide gel electrophoresis and in 55 samples by a group A-specific enzyme-linked immunosorbent assay (ELISA). All samples which were positive by polyacrylamide gel electrophoresis and negative by ELISA had elicited atypical electropherotypes resembling those of group B or C. ELISA-positive samples showing genome rearrangements were also detected (R.C. Bellinzoni, N.M. Mattion, O.R. Burrone, S.A. González, J.L. La Torre, and E.A. Scodeller, J. Clin. Microbiol. 25:952-954, 1987; N.M. Mattion, S.A. González, O.R. Burrone, R.C. Bellinzoni, J.L. La Torre, and E.A. Scodeller, J. Gen. Virol. 69:695-698, 1988). By subgrouping with monoclonal antibodies, it was found that of 32 positive samples, 13 belonged to subgroup I, 2 belonged to subgroup II, 2 samples had both specificities, and 15 samples were neither subgroup I nor subgroup II (non-I/II). In addition, 10 samples were adapted to grow in tissue culture, cloned, and serotyped by means of neutralization assays. Two samples were classified as serotype 5, and none of them were classified as serotype 4. The other strains showed only a one-way relationship with serotype 5 and can be tentatively classified as new porcine serotypes. Two samples with rearranged genomes had a one-way relationship with antiserum to human strain 69M, which displays a supershort electropherotype and was classified as a new human serotype (S. Matsuno, A. Hasegawa, A. Mukoyama, and S. Inouye, J. Virol. 54:623-624, 1985). At one farm, similar rearranged strains were detected during three successive years. Serotype changes were found between the isolates of the first and the second year, suggesting that a high degree of antigenic variability went on during continuous circulation of these strains in the field.

Isolation of group A swine rotaviruses displaying atypical electropherotypes.

Swine rotaviruses displaying distinctive electropherotypes were isolated from the feces of diarrheic piglets in two swine herds in the province of Buenos Aires, Argentina. In one case all samples isolated showed abnormal electropherotypes. All samples were classified as group A reactive when assayed by an enzyme-linked immunosorbent assay. Three samples from this herd were adapted to grow in tissue culture. The electrophoretic pattern of the genomic RNA as well as the group A reactivity of one of these viruses was retained after cloning in MA-104 cells. In the other pig unit were found samples displaying both classical and abnormal electropherotypes. These viruses were also positive in the enzyme-linked immunosorbent assay; however, since they could not be adapted to grow in tissue culture, this classification must be considered tentative. The abnormal electropherotype exhibited by these pig viruses strongly resembles those of human origin called super short.