Effects of deletions of kelch-like genes on cowpox virus biological properties Article First Online: 14 April 2005 Received: 17 November 2004 Accepted: 17 February 2005 DOI:
Cite this article as: Kochneva, G., Kolosova, I., Maksyutova, T. et al. Arch Virol (2005) 150: 1857. doi:10.1007/s00705-005-0530-0 Summary.
Cowpox virus (CPXV) strain GRI-90 contains six genes encoding
kelch-like proteins. All six proteins contain both, the N-terminal BTB domain and the C-terminal kelch domain. We constructed mutant variants of a CPXV strain with targeted deletions of one to four genes of the kelch family, namely D11L, C18L, G3L, and A57R. As kelch genes are located in terminal variable regions of the CPXV genome, we studied the relationship of these genes with integral biological characteristics such as virulence, host range, reproduction in vitro and in ovo (in chicken embryos). It was demonstrated that the following effects occurred in a gene dose dependent manner with an increase of the number of genes deleted: (1) range of sensitive cells altered – deletion mutants lacking three genes displayed a considerably decreased ability to reproduce in MDCK cells; mutants lacking four genes lost this ability completely; (2) analysis of pocks formed by mutants with deletion of three and four kelch-like genes on chorioallantoic membranes of chicken embryos demonstrated that pock size and virus yield were significantly decreased; (3) light microscopic analysis of the pocks revealed impaired proliferation and reduced vascularisation in the pock region. More alterations were detected by electron microscopic analysis: the reproduction of mutants results in a reduction of the number of mature virions formed, and in many cells this process was arrested at the stage of assembly of immature virions; and (4) the evaluation of LD 50 and body weight loss in BALB/c mice infected intranasally with CPXVs revealed a reduction of the virulence of the deletion mutants, which became statistically significant when four kelch-like genes were excised. References Ahmad, KF, Engel, CK, Prive, GG 1998 Crystal structure of the BTB domain from PZLF. Proc Natl Acad Sci USA 95 12123 12128 CrossRef PubMed Google Scholar Chen, N, Danila, MI, Feng, Z, Buller, RM, Wang, C, Han, X, Lefkowitz, EJ, Upton, C 2003 The genomic sequence of ectromelia virus, the causative agent of mousepox. Virology 317 165 186 CrossRef PubMed Google Scholar Coulter, HD 1976 Rapid and improved method for embedding biological tissues in Epon 812 and Araldit 502. J Ultrastr Res 20 346 355 CrossRef Google Scholar de Miranda, MP, Reading, PC, Tscharke, DC, Murphy, BJ, Smith, GL 2003 The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model. J Gen Virol 84 2459 2471 CrossRef PubMed Google Scholar Falkner, FG, Moss, B 1990 Transient dominant selection of recombinant vaccinia viruses. J Virol 64 3108 3111 PubMed Google Scholar Ichihashi, Y, Matsumoto, S, Dales, S 1971 Biogenesis of poxviruses: role of A-type inclusions and host cell membranes in virus dissemination. Virology 46 507 532 CrossRef PubMed Google Scholar Kochneva, GV, Urmanov, IH, Ryabchikova, EI, Streltsov, VV, Serpinsky, OI 1994 Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence. Virus Res 34 49 61 CrossRef PubMed Google Scholar Kolosova, IV, Seregin, SV, Kochneva, GV, Ryabchikova, EI, Bessmel’tseva, EV, Babkina, IN, Solenova, TE, Babkin, IV, Shchelkunov, SN 2003 Orthopoxvirus genes for kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions. Mol Biol (in Russian) 37 585 594 Google Scholar Kotwal, GJ, Moss, B 1988 Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 167 524 537 PubMed Google Scholar Marennikova, SS, Gashnikov, PV, Zhukova, OA, Ryabchikova, EI, Streltsov, VV, Ryazankina, OI, Chekunova, EV, Yanova, NN, Shchelkunov, SN 1996 Biotype and genetic characterization of the isolate of cowpox virus having caused infection in a child. Zh Mikrobiol (in Russian) 4 6 10 Google Scholar
Marennikova SS, Shchelkunov SN (1998) Orthopoxviruses Pathogenic for humans. KMK Scientific Press, Moscow
Martinez, MJ, Bray, MP, Huggins, JW 2000 A mouse model of aerosol-transmitted orthopoxviral disease: morphology of experimental aerosol-transmitted orthopoxviral disease in a cowpox virus-BALB/c mouse system. Arch Pathol Lab Med 124 362 377 PubMed Google Scholar Massung, RF, Liu, LI, Qi, J, Knight, JC, Yuran, TE, Kerlavage, AR, Parsons, JM, Venter, JC, Esposito, JJ 1994 Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology 201 215 240 CrossRef PubMed Google Scholar Perkus, ME, Goebel, SJ, Davis, SW, Johnson, GP, Norton, EK, Paoletti, E 1991 Deletion of 55 open reading frames from the termini of vaccinia virus. Virology 180 406 410 CrossRef PubMed Google Scholar Robinson, DN, Cooley, L 1997 Drosophila kelch is an oligomeric ring canal actin organizer. J Cell Biol 138 799 810 CrossRef PubMed Google Scholar Ryabchikova, EI, Strel’tsov, VV, Petrov, VS 1990 Submicroscopic modifications of chorion-allantoic membrane of chicken embryos infected with vaccinia virus. Problems Virology (in Russian) 6 506 509 Google Scholar Ryazankina, OI, Tumanova, OYu, Kolosova, IV, Safronov, PF, Kablova, GV, Ryazankin, IA, Shchelkunov, SN 2000 Structural-functional organization of cowpox virus GRI-90 genome. I. Isolation of clones of DNA fragments of complete cowpox virus genome. Mol Biol (in Russian) 34 160 168 Google Scholar Safronov, PF, Totmenin, AV, Ryazankina, OI, Shchelkunov, SN 1999 Structure-activity organization of the cowpox strain GRI-90 viral genome. III. Functional characteristics of the left species-specific region of the genome. Mol Biol (in Russian) 33 303 313 Google Scholar Shchelkunov, SN, Blinov, VM, Resenchuk, SM, Totmenin, AV, Olenina, LV, Chirikova, GB, Sandakhchiev, LS 1994 Analysis of the nucleotide sequence of 53 kpb from the right terminus of the genome of variola major virus strain India-1967. Virus Res 34 207 236 CrossRef PubMed Google Scholar Shchelkunov, SN, Safronov, PF, Totmenin, AV, Petrov, NA, Ryazankina, OI, Gutorov, VV, Kotwal, GJ 1998 The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243 432 460 CrossRef PubMed Google Scholar Shchelkunov, SN, Totmenin, AV, Loparev, VN, Safronov, PF, Gutorov, VV, Chizhikov, VE, Knight, JC, Parsons, JM, Massung, RF, Esposito, JJ 2000 Alastrim smallpox variola minor virus genome DNA sequences. Virology 266 361 386 CrossRef PubMed Google Scholar Shchelkunov, S, Totmenin, A, Kolosova, I 2002 Species-specific differences in organization of orthopoxvirus kelch-like proteins. Virus Genes 24 157 162 CrossRef PubMed Google Scholar Shchelkunov, SN, Totmenin, AV, Safronov, PF, Mikheev, MV, Gutorov, VV, Ryazankina, OI, Petrov, NA, Babkin, IV, Uvarova, EA, Sandakhchiev, LS, Sisler, JR, Esposito, JJ, Damon, IK, Jahrling, PB, Moss, B 2002 Analysis of the monkeypox virus genome. Virology 297 172 194 CrossRef PubMed Google Scholar Zollman, S, Godt, D, Prive, GG, Couderc, JL, Laski, FA 1994 The BTB domain found primarily in zing finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci USA 91 10717 10721 PubMed Google Scholar Copyright information
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