Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.The human papillomaviruses (HPVs) are small DNA tumor viruses (20). To date, more than 100 different types have been identified (12). Some of these HPV types, termed high-risk types, are associated with development of cancer of the uterine cervix, one of the most common cancers in women worldwide (35,50). HPV type 16 (HPV-16) is the most common high-risk type (51). The HPV genome can be divided into an early region and a late region, followed by the proximal early (pAE) and the distal late (pAL) polyadenylation signals, respectively (Fig. 1A) (4). A polyadenylation signal consists of an AA UAAA sequence located 10 to 30 nucleotides (nt) upstream of the cleavage site and a degenerate GU-rich sequence element about 30 nucleotides downstream of the cleavage site (45). Some polyadenylation signals are followed by G-rich downstream elements (2, 3). The weak binding of cleavage/polyadenylation specificity factor (CPSF) to the AAUAAA motif is enhanced by CStF binding to the downstream GU-rich element (45). Recently, it was shown that U-rich upstream polyadenylation elements interact with hFip-1, an integral part of CPSF (21). In the early stage of the viral life cycle, all viral transcripts are polyadenylated at the pAE, whereas both the pAE and the pAL are used in the late, productive stage of the infection (26). In response to differentiation of the HPV-infected cell, the use of the pAE is down regulated, resulting in read-through into the late region and polyadenylation of the late transcripts at the pAL. Efficient polyadenylation at the pAE is an absolute requirement for early...