Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles

We have previously published a detailed transcription map of Aleutian mink disease parvovirus (ADV) and proposed a model for the translation of the two virion structural proteins (VP1 and VP2) and three nonstructural proteins (NS-1, NS-2, and NS-3) (S. Alexandersen, M. E. Bloom, and S. Perryman, J. Virol. 62:3684-3994, 1988). To verify and further characterize this model, we cloned the predicted open reading frames for NS-1, NS-2, NS-3, VP1-VP2, and VP2 alone into a recombinant baculovirus and expressed them in Sf9 insect cells. Expression of VP1-VP2 or VP2 alone in cDNA and in the genomic form was achieved. The expressed proteins had molecular weights similar to those of the corresponding proteins of wild-type ADV-G, although the ratio of VP1 to VP2 was altered. The recombinant baculovirus-expressed ADV VP1 and VP2 showed nuclear localization in Sf9 cells and were able to form particles indistinguishable, by electron microscopy, from wild-type virus. The large nonstructural protein, NS-1, showed predominantly nuclear localization in Sf9 cells when analyzed by immunofluorescence and had a molecular weight similar to that of wild-type ADV NS-1. Moreover, expression of NS-1 in Sf9 cells caused a change in morphology of the cells and resulted in 10-times-lower titers of recombinant baculovirus during infection, suggesting a cytostatic or cytotoxic action of this protein. The smaller NS-2 gene product seems to be located in the cytoplasm. When analyzed by Western immunoblotting, NS-2 comigrated with an approximately 16-kDa band seen in lysates of ADV-infected feline kidney cells. The putative NS-3 gene product exhibited a diffuse distribution in Sf9 cells and had a molecular weight of approximately 10,000. All of the expressed ADV-encoded proteins were recognized by sera from ADV-infected mink. Thus, expression of ADV cDNAs allowed assignment of the different mRNAs to the viral proteins observed during ADV infection in cell culture and supported our previously proposed ADV transcriptional and translational scheme. Moreover, the production of structural proteins from a full-length NS-2 mRNA may add to the repertoire of parvovirus gene expression.

Section: Discussionmentioning

confidence: 99%

Expression of Aleutian mink disease parvovirus proteins in a baculovirus vector system

Christensen

Storgaard

Bloch

et al. 1993

“…Virions were partially purified from lysates of ADV-G-infected CRFK cells that had been freeze-thawed and sonicated. After clarification and extraction of lipids with chloroform, virus-containing suspensions were layered over 40% sucrose in PBS and ultracentrifuged at 25,000 rpm overnight at 4ЊC in a Beckman SW41 rotor (Beckman Instruments, Fullerton, Calif.) (23,59). The pellets were resuspended in PBS by sonication and passed through a 100-nm-pore-size filter to minimize virus aggregates.…”

Section: Construction Of Adv Capsid Protein Expression Plasmidsmentioning

confidence: 99%

“…The pellets were resuspended in PBS by sonication and passed through a 100-nm-pore-size filter to minimize virus aggregates. An identical procedure was used to obtain self-assembled empty capsids from Sf9 cells infected with the recombinant baculovirus Ac-ADV-1 (23,59).…”

Section: Construction Of Adv Capsid Protein Expression Plasmidsmentioning

confidence: 99%

See 1 more Smart Citation

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions

Bloom

Martin

Oie

et al. 1997

Self Cite

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.

“…Native ADV virions contain 60 protein subunits, with VP1 and VP2 comprising, respectively, about 10 and 90% of the total. ADV capsid proteins expressed in recombinant vaccinia or baculovirus self-assemble into particles which appear to have the physicochemical and antigenic properties of native virions, though the ratio of VP1 to VP2 in the expressed capsids is reversed (26,27,68). Expressed VP2, the major capsid protein, can self-assemble into native-like capsids in the absence of VP1 (26), as has also been observed for other parvoviruses in mammalian cells (64).…”

mentioning

confidence: 70%

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

McKenna

Olson

Chipman

et al. 1999

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 Å resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 Å and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related β-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.