A polypyrimidine tract facilitates the expression of Kaposi's sarcoma-associated herpesvirus vFLIP through an internal ribosome entry site.
We have identified a novel internal ribosome entry site (IRES) within a latently expressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene (vCyclin) that controls the expression of a downstream open reading frame encoding an inhibitor of apoptosis (vFLIP). This IRES is the first such element to be identified in a DNA virus and may represent a novel mechanism through which this virus controls gene expression. We have used a dual luciferase reporter assay to identify important sequence elements essential for the activity of the IRES. A sequence of 32 nucleotides incorporating a polypyrimidine tract (PPT) was found to be required for the proper functioning of the IRES. We also show, using an electrophoretic mobility shift assay (EMSA), that proteins specific to a KSHV-infected cell line (BCP-1) but not a KSHV-negative cell line (HEK293) were able to form complexes with the IRES. By using an in vitro RNA binding assay, the cellular polypyrimidine tract binding protein (PTB, hnRNP-I) was found to bind to the IRES RNA. These results suggest that the interaction of PTB with the PPT may contribute to the correct functioning of the KSHV IRES in infected cells.
University of Edinburgh, Centre for Infectious Diseases, Summerhall, Edinburgh EH9 1QH, UK.
Lara
L
Bieleski
University of Edinburgh, Centre for Infectious Diseases, Summerhall, Edinburgh EH9 1QH, UK.
Clemence
C
Hindley
University of Edinburgh, Centre for Infectious Diseases, Summerhall, Edinburgh EH9 1QH, UK.
Simon J
SJ
Talbot
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011743:Pyrimidines; D011991:Receptors, Virus / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012270:Ribosomes / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D011743:Pyrimidines; D011991:Receptors, Virus; D011994:Recombinant Proteins; D014764:Viral Proteins; C481275:viral FLIP protein, Human herpesvirus 8; D008156:Luciferases