The agnogene of the human polyomavirus BK is expressed

Abstract

Primate polyomavirus genomes all contain an open reading frame at the 5' end of the late coding region called the agnogene. A simian virus 40 agnoprotein with unknown functions has previously been demonstrated. We now show that a BK virus agnoprotein appears in the perinuclear area and cytoplasm late in the infectious cycle. It is phosphorylated in vivo and coimmunoprecipitates with a subset of host cell proteins.

FIG. 1

Western blot analysis of BKV agnoprotein (Agno) synthesis in HUV-EC-C. (A) Time course of agnoprotein synthesis, detected by using purified agnoprotein antibodies. (B) Extracts from cells transfected with an agnoprotein expression plasmid (lane 2), compared with mock-transfected (lane 1) and 48-h-p.i. BKV-infected (lane 3) cells. The positions of molecular mass markers are indicated.

FIG. 2

Subcellular localization of the BKV agnoprotein (Agno). Shown are immunoperoxidase-stained BKV-infected HUV-EC-C cells at 72 h p.i. Purified antiagnoprotein antibodies were used.

FIG. 3

In vivo phosphorylation of BKV agnoprotein. (A) Immunoblot of cell lysates from mock-infected (lane 1) and BKV-infected (lane 2) HUV-EC-C cells after metabolic labelling with ³²P. The lysates were subjected to immunoprecipitation with purified antiagnoprotein antibodies prior to SDS-PAGE and Western blotting. The elution positions of the immunoglobin heavy chain (Ig) and agnoprotein (Agno) are indicated by arrows. (B) PhosphorImager analysis of the blot in panel A, showing phosphorylated immunoprecipitated proteins. The positions of molecular mass markers are indicated.

FIG. 4

Coimmunoprecipitation of higher-molecular-weight proteins of cellular origin with BKV agnoprotein. BKV- or mock-infected HUV-EC-C cells were metabolically labelled with [³H]leucine, and then cell lysates were immunoprecipitated with purified antiagnoprotein antibodies or unrelated antibodies and subjected to SDS-PAGE and fluorography. Lanes: 1, lysates from mock-infected cells immunoprecipitated with antiagnoprotein antibodies; 2, lysates from BKV-infected cells immunoprecipitated with antiagnoprotein antibodies; 3, lysates from BKV-infected cells immunoprecipitated with unrelated antibodies; 4, lysates from radiolabelled, mock-infected HUV-EC-C cells; 5, lysates from mock-infected [³H]labelled HUV-EC-C cells that were incubated with lysates from unlabelled, BKV-infected HUV-EC-C cells prior to immunoprecipitation. The positions of molecular mass markers are indicated.