Human papillomavirus genotype 31 does not express detectable microRNA levels during latent or productive virus replication

Abstract

It has recently become clear that several pathogenic DNA viruses express virally encoded microRNAs in infected cells. In particular, numerous microRNAs have been identified in a range of human and animal herpesviruses, and individual microRNAs have also been identified in members of the polyoma- and adenovirus families. Although their functions remain largely unknown, it seems likely that these viral microRNAs play an important role in viral replication in vivo. Here we present an analysis of the microRNAs expressed in human cells during the latent and productive phases of the human papillomavirus genotype 31 (HPV31) replication cycle. Although over 500 cellular microRNAs were cloned and identified, not a single HPV31-specific microRNA was obtained. We therefore concluded that HPV31, and possibly human papillomaviruses in general, does not express viral microRNAs.

FIG. 1.

Northern and Southern analyses of undifferentiated and methylcellulose-differentiated HPV31-positive LKP1 cells. LKP1 cells were harvested for analysis following growth in undifferentiated monolayer cultures (0 h) or following differentiation in 1.5% methylcellulose for 24 and 48 h. (A) Southern analysis of total LKP1 DNA showing HPV31 amplification following suspension in methylcellulose. Total DNA was sheared prior to being loaded into the gel and analyzed as previously described (8). Supercoiled (S), open circular DNA (O), and linear (L) forms of the HPV31 genome are indicated. (B) Northern analysis of total RNAs from undifferentiated and differentiated LKP1 cells showing induction of late gene expression following suspension in methylcellulose for 24 and 48 h. Early transcripts include E6-E7-E1^E4-E5 and E6*-E7-E1^E4-E5, and late transcripts include E1^E4-E5 and E1-E4-E5. Total RNA was isolated using a mirVANA isolation kit from Ambion Inc. (Austin, TX). The RNAs from the 24- and 48-h methylcellulose time points were combined for the miRNA cloning analysis.

FIG. 2.

Origins of 18- to 25-nt sequences cloned from undifferentiated (A) and differentiated (B) LKP1 cells. RNAs of between 18 and 25 nt were cDNA cloned from total RNA preparations derived from undifferentiated and differentiated LKP1 cells, as shown in Fig. 1B. The resultant short cDNAs were concatenated, cloned, and sequenced. The origin of each of the 339 distinct sequences obtained from undifferentiated LKP1 cells (A) and the 359 sequences obtained from differentiated cells (B) was deduced by computer analysis and is shown. No HPV31-derived sequences of any kind were obtained.