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Journal of Virology, September 1999, p. 7907-7911, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumavirus

Klaus-Ingmar Pfrepper,1 Martin Löchelt,1 Hans-Richard Rackwitz,2 Martina Schnölzer,3 Hans Heid,2 and Rolf M. Flügel1,*

Department of Retroviral Gene Expression, Research Program Applied Tumor Virology,1 Department of Cell Biology, Research Program Cell Differentiation and Carcinogenesis,2 and Protein Analysis Facility,3 German Cancer Research Center, 69009 Heidelberg, Federal Republic of Germany

Received 4 November 1998/Accepted 11 June 1999

Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.

* Corresponding author. Mailing address: Abteilung Retroviral Gene Expression, Applied Tumor Virology, DKFZ, INF 242, 69009 Heidelberg, Federal Republic of Germany. Phone: 49-6221-424611. Fax: 49-6221-424865. E-mail: r.m.fluegel{at}dkfz-heidelberg.de.

Journal of Virology, September 1999, p. 7907-7911, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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