Little is known about the nature of poxvirus proteins involved in the host immune response. Screening a lambda gt11 expression library of genomic rabbit poxvirus DNA with hyperimmune rabbit anti-vaccinia virus serum and selection of monospecific antibodies identified a highly antigenic viral protein of about 39,000 molecular weight (39K protein). The same-size protein of vaccinia virus was also identified with a monoclonal antibody (MAb B6) obtained from hybridomas generated after fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Structural analysis revealed that the 39K protein is an acidic polypeptide, that it can exist in two molecular forms because of intramolecular disulfide linkages, and that it is part of the virus core. This protein shares antigenic determinants with a cytoplasmic component(s) from uninfected cells. Functional studies revealed that the 39K protein is synthesized at late times postinfection and appears to be required for virus assembly. This protein is highly conserved in members of the Orthopoxvirus group, but in cowpox virus, a 41K virion protein was specifically recognized by antibodies that reacted against the vaccinia virus 39K protein. Significantly, during long-term passages of Friend erythroleukemia cells persistently infected with vaccinia virus, some virus mutants were found to increase or decrease by about 2 kilodaltons the size of the 39K protein. Mapping analysis localized sequences encoding the 39K protein in a rifampin-sensitive gene cluster between the two major core-associated viral polypeptides, 4a and 4b. The fact that the 39K core protein of vaccinia virus elicits strong humoral immune response, induces antibodies that react against a host component(s), and is subjected to genetic variability suggests that this protein has important biological functions.

Vaccinia virus (W) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gtll expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of W, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins. * Corresponding author. MATERIALS AND METHODS Materials. Restriction endonucleases, modifying enzymes, isopropyl- ,-D-thiogalactopyranoside (IPTG), and protein A were obtained from Bethesda Research Laboratories, Inc., New England Biolabs, or Boehringer Mannheim Biochemicals. The radioisotopes [3H]thymidine, [35S]methionine, 125I, and [32P]dATP were from Amersham or ICN. Goat anti-rabbit peroxidase and goat anti-human peroxidase were from Organon Teknika, Cappel. Cloning, mapping, and DNA sequence analysis. DNA manipulations were carried out as previously described (31). DNA fragments obtained by digestion of viral and phage 386

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Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA.

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