High-risk human papillomaviruses (HPVs) link their life cycle to epithelial differentiation and require activation of DNA damage pathways for efficient replication. HPVs modulate the expression of cellular transcription factors, as well as cellular microRNAs (miRNAs) to control these activities. One miRNA that has been reported to be repressed in HPV-positive cancers of the cervix and oropharynx is miR-424. Our studies show that miR-424 levels are suppressed in cell lines that stably maintain HPV 31 or 16 episomes, as well as cervical cancer lines that contain integrated genomes such as SiHa. Introduction of expression vectors for miR-424 reduced both the levels of HPV genomes in undifferentiated cells and amplification upon differentiation. Our studies show that the levels of two putative targets of miR-424 that function in DNA damage repair, CHK1 and Wee1, are suppressed in HPV-positive cells, providing an explanation for why this microRNA is targeted in HPV-positive cells.IMPORTANCE We describe here for the first time a critical role for miR-424 in the regulation of HPV replication. HPV E6 and E7 proteins suppress the levels of miR-424, and this is important for controlling the levels of CHK1, which plays a central role in viral replication.KEYWORDS amplification, HPV, microRNA, DNA damage repair, CHK1, check point H uman papillomaviruses (HPVs) are the causative agents of cervical and other anogenital cancers (1). HPVs are classified as either high-risk types (such as HPV16, -18, -31, and -35), which are associated with the development of cancers of the anogenital tract and oropharynx or low-risk groups (such as HPV6 and HPV11) that are infrequently detected in malignancies. HPV virions infect cells in the basal layer of stratified epithelia to establish persistent infections. In infected basal cell cells, genomes are maintained as low-copy episomes that replicate in synchrony with cellular chromosomes. Productive viral replication or amplification, however, occurs only upon epithelial differentiation in suprabasal layers (2, 3). High-risk HPVs encode two major oncoproteins, E6 and E7, that play critical roles in cell transformation and provide important regulatory functions during the differentiation-dependent life cycle. One major target of E6 is the E3 ligase E6-associated protein E6AP, which forms trimeric complexes with p53, resulting in its rapid turnover (4-6). Similarly, E7 binds and induces the degradation of pRb, which results in the constitutive activation of E2F family members, leading to altered cell cycle control and differentiation (7,8). Additional important targets of these viral proteins include the ataxia telangiectasia-mutated (ATM) and the ATM and Rad3-related (ATR) DNA repair pathways that play critical roles in HPV replication (9, 10). E6 and E7 regulate these pathways by altering the expression