A-type Inclusion Body Protein Research Articles

More cell culture passaged Camelpox virus sequences found resembling those of vaccinia virus.

Background:Camelpox is the most infectious and economically important disease of camelids that causes significant morbidity and mortality rates. Several live attenuated vaccines against Camelpox virus (CMLV) are produced worldwide by passaging field isolates in cell culture. Sequence of a high passage Saudi isolate of CMLV was previously found closely resembled Vaccinia virus (VACV). Aim:To determine whether other high cell culture passage CMLV isolates are genetically resemble VACV and further to explore the possible mechanism of the resemblance.Methods:We performed polymerase chain reaction and DNA sequence analysis of A-type inclusion body protein (ATIP), L1R, and open reading frame (ORF) 185 genes on different cell culture passage levels of a field isolate, two high passage vaccines, wild-type, and reference strains of CMLV.Results:We demonstrate that additional two high passage attenuated vaccine candidate from Sudan and UAE likewise contain sequences resembling VACV more than CMLV. Furthermore, sequence analysis of the ATIP gene of selected virus passages in cell culture revealed that the shift to VACV-like occurred between passage 11 and 20 and up to the 10th passage the genome still resembles wild-type virus. This observation was further confirmed by recombination analysis which indicated recombination events at ATIP and ORF185 genes occurred at higher passages.Conclusion:We confirmed that the cell culture passage CMLV turns to resemble VACV after cell culture passage and concluded that the resemblance may not be a result of contamination or misidentification as previously thought but could be due to recombination events that occurred during the passage process.

Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein. Since new-generation smallpox vaccines such as MVA will require extensive comparison to traditional smallpox vaccines in animal models of immunogenicity and protection, we compared the vaccinia virus antigens recognized by VIG to those recognized by sera from Dryvax and MVA immunized mice. The humoral immune response in immunized mice is qualitatively similar to that in humans.

Specific detection of monkeypox virus by polymerase chain reaction

The open reading frame coding for the A-type inclusion body protein (ATI) of monkeypox virus (MPV) was identified and sequenced for two strains. Nucleotide sequence comparison revealed 72–95.3% homology with the reported open reading frame sequences of the ATIs of other orthopoxvirus species, such as variola, vaccinia, cowpox, ectromelia, and camelpox viruses. Each MPV strain contained an 8-bp deletion, which caused a frameshift that introduced a premature stop in the open reading frame at base 2091 relative to the ATI open reading frame of cowpox virus strain Brighton. The sequences enabled a primer pair to be designed that flanked the deletion and specifically amplified a 601-bp fragment that identified and differentiated 19 MPV strains examined from five other Old World orthopoxvirus species examined. The specificity was confirmed by cleavage of the 19 MPV strain amplicons with BglII, which produced three subfragments of expected sized, based on the determined MPV sequences.

Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses

Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.

Constructions of vaccinia virus A-type inclusion body protein, tandemly repeated mutant 7.5 kDa protein, and hemagglutinin gene promoters support high levels of expression.

We devised vaccinia virus (VV)-based vector systems that support higher levels of expression of cloned genes in the early and late phases of the infection cycle than reported previously. Enhanced expression can be obtained by combining the promoter of the A-type inclusion body protein gene, the mutated early region of the 7.5-kDa gene promoter (7.5-kDa promoter), and the promoter of the hemagglutinin (HA) gene. One construct produced 60 micrograms/10(6) cells of chloramphenicol acetyltransferase (CAT), equivalent to 10-18% of total cell protein. Another construct produced about seven times more CAT during the early phase than the amount synthesized under the control of the 7.5-kDa promoter alone. The envelope proteins of human immunodeficiency virus type I synthesized during the early phase of infection were more active as immunogens than these proteins synthesized during the late phase, regardless of the amounts produced.

Characterization of the gene encoding the A-type inclusion body protein of mousepox virus.

The gene of the mousepox virus strain MP-1 coding for the protein of A-type inclusions was identified and sequenced. The gene maps in the HindIII-A fragment at the same position as the cowpox-virus Brighton 160 kD gene (1) and the closely related 94 kD LS gene of vaccinia virus strain Western Reserve (2,3). The encoded protein consists of 1049 amino acids with a predicted molecular weight of 122.5 kD, which corresponds well to the apparent molecular weight of 130 kD estimated after polyacrylamide gel electrophoresis and Western blotting. The protein is a characteristic feature of mousepox virus and could be demonstrated for five independently isolated strains. The MP-1 ATI protein displays a similarity of 82.3% to the cowpox virus Brighton 160 kD protein, the first 340 amino acids being almost identical. However, two major deletions of 55 and 92 amino acids were observed from amino acid positions 671 to 732 and 794 to 885, respectively. Furthermore, the C-terminal residues (position 998-1049 of the MP-1 sequence) did not match at satisfactory levels with cowpox nor with vaccinia virus sequences. According to Western blot and protein alignment data as well as hydropathy predictions, the 130 and 160 kD proteins closely resemble each other, indicating similar structure and function.

Characterization of the gene encoding the A-type inclusion protein of camelpox virus and sequence comparison with other orthopoxviruses

A gene was identified in camelpox virus strain CP-1 that is similar to the 160K gene of cowpox virus strain Brighton (BR) that encodes the A-type inclusion body protein (ATIP). The CP-1 gene was mapped, sequenced, and the presence of the ATIP-specific mRNA was demonstrated. The open reading frame [2178 nucleotides (nt)] was found at a similar position in the CP genome as the one reported for the cowpox virus 160K ATI gene. DNA sequence comparison revealed a deletion of two adjacent adenine residues relative to cowpox virus BR, generating a reading frame shift accompanied by the formation of a translational stop codon. An identical deletion has been described for vaccinia virus strain Western Reserve. The DNA sequence of the corresponding region of monkeypox virus strain Copenhagen revealed a deletion leading to a putative stop codon 75 nt upstream of the same stop codons in the camelpox and vaccinia virus genes. These findings are consistent with the expression of truncated ATIPs, of 94K in vaccinia and camelpox viruses and of 92K in monkeypox virus. In addition, a deletion of 789 bp could be localized downstream of the ATI open reading frame in camelpox virus isolates of different origin. This causes the transcription of a shortened ATI-specific mRNA (3.7 kb) relative to vaccinia and cowpox viruses (both 4.5 kb). The similarity observed in ATIP-encoding and flanking sequences might suggest that vaccinia and camelpox viruses are descended from a common ancestor.

The predicted amino acid sequence of the spheroidin protein from Amsacta moorei entomopoxvirus: lack of homology between major occlusion body proteins of different poxviruses.

Entomopoxviruses replicate in the cytoplasm of insect cells and characteristically produce occlusion bodies which serve to protect the virion from the environment; the major component of these bodies is a protein called spheroidin. We have previously identified and sequenced the gene encoding the major occlusion body protein of eastern spruce budworm (Choristoneura biennis) entomopoxvirus (CbEPV) and found it to encode a 47K polypeptide which aggregates due to the formation of intermolecular disulphide bonds. In this publication we demonstrate that the insect poxvirus of Amsacta moorei produces spheroidin with a unit Mr of 114.8K. The gene for this protein was cloned and sequenced, and the predicted polypeptide was demonstrated to contain 38 cysteine residues, a leucine zipper for possible protein-protein interactions and 14 potential Asn-linked glycosylation sites. Other than possessing a large number of sulphydryl groups, this protein showed no homology to its analogue found in cells infected with CbEPV. Antibodies directed against occlusion body proteins of the two viruses also failed to cross-react significantly on Western blots. In addition, nucleic acid probes prepared from the two different genes did not cross-hybridize on Southern blots of genomic DNA prepared from the viruses. Finally, the occlusion body proteins from the two insect viruses were compared with the A-type inclusion body protein of cowpox virus. Again, little homology between these proteins was evident, with the exception of a generally high cysteine content and a similarity between their late gene promoters. We conclude that the major occlusion body proteins of different poxviruses possess diverse primary structures, but all are capable of yielding large aggregates through the formation of disulphide bonds.

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.

High-level synthesis of biologically active reovirus protein σ1 in a mammalian expression vector system

The reovirus cell-attachment protein, which modulates tissue tropism and the nature of the antiviral immune response, is protein σ1. This protein is present in reovirus particles in the form of 12 tetramers anchored in the projections or spikes. It is formed in infected cells in very small amounts only. In order to produce larger amounts of it that are necessary for studying its properties in detail, we have cloned it into a mammalian expression vector system that consists of the very strong cowpox virus A-type inclusion body protein gene promoter inserted into the vaccinia virus genome. In cells infected with the resultant recombinant vaccina virus, protein σ1 is formed in large amounts (2–3 mg/10 9 cells). We have isolated the native protein and shown that it exists as tetramers that possess cell-binding activity, hemagglutinating activity, and functional epitopes recognized by both polyvalent antisera and monoclonal antibodies.