The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels

^*Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow, G11 5JR, Scotland, United Kingdom; and ^†Department of Biological Sciences, Columbia University, New York, NY 10027

Communicated by Robert Louis Sinsheimer, University of California, Santa Barbara, CA (received for review November 22, 1999)

Abstract

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3′ untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

Footnotes

↵ ‡ To whom reprint requests should be addressed. E-mail: b.clements{at}vir.gla.ac.uk.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.070049097.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.070049097
Abbreviations:

HPV16,

human papillomavirus type 16;

NRE,

negative regulatory element;

UTR,

untranslated region;

U2AF65,

U2 auxiliary splicing factor 65-kDa subunit;

CstF-64,

cleavage stimulation factor 64-kDa subunit;

poly(A),

polyadenylation;

PMA,

phorbol-12-myristate-13-acetate;

EMSA,

electrophoretic mobility-shift assay;

GST,

glutathione S-transferase;

RBD,

RNA-binding domain;

CAT,

chloramphenicol acetyltransferase