MicroRNAs (miRNAs) play important roles in cancer development. By cloning and sequencing of a HPV16+ CaSki cell small RNA library, we isolated 174 miRNAs (including the novel miR-193c) which could be grouped into 46 different miRNA species, with miR-21, miR-24, miR-27a, and miR-205 being most abundant. We chose for further study 10 miRNAs according to their cloning frequency and associated their levels in 10 cervical cancer- or cervical intraepithelial neoplasia-derived cell lines. No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome. All cell lines examined contained no detectable miR-143 and miR-145. HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture, including expression of miR-143 and miR-145. This suggests a correlation between miRNA expression and tissue differentiation. Using miRNA array analyses for age-matched normal cervix and cervical cancer tissues, in combination with northern blot verification, we identified significantly deregulated miRNAs in cervical cancer tissues, with miR-126, miR-143, and miR-145 downregulation and miR-15b, miR-16, miR-146a, and miR-155 upregulation. Functional studies showed that both miR-143 and miR-145 are suppressive to cell growth. When introduced into cell lines, miR-146a was found to promote cell proliferation. Collectively, our data indicate that downregulation of miR-143 and miR-145 and upregulation of miR-146a play a role in cervical carcinogenesis.

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.HSV ͉ latency-associated transcript ͉ latency ͉ reactivation ͉ human H erpes simplex virus 1 and 2 (HSV-1 and HSV-2) are closely related human herpes viruses. HSV-1 and HSV-2 establish lifelong incurable latency in and reactivate preferentially from trigeminal ganglia and dorsal root ganglia to cause oro-facial and genital herpes, respectively. Although infections are usually mild, these viruses can cause severe disease including encephalitis and neonatal herpes, and HSV-2 infection is a risk factor for HIV acquisition. The only readily detectable viral transcript during latency of both HSV-1 and HSV-2 is the noncoding latency-associated transcript (LAT), which is transcribed from within the long repeats of the viral genome ( Fig. 1) (1). A Ϸ8-kb primary LAT is spliced, yielding a stable Ϸ2-kb LAT intron (2). Deletion of the LAT promoter in both HSV-1 and HSV-2 reduces the efficiency of reactivation (3-5), and substitution of HSV-1 LAT for native HSV-2 LAT sequences confers an HSV-1 reactivation phenotype (6). The HSV-1 LAT is currently believed to act in part by increasing the establishment or maintenance of latency, likely via an effect on the survival of acutely infected neurons (7), which may be mediated by inhibition of apoptosis in infected neurons (8). The molecular function of HSV-2 LAT remains largely unknown.miRNAs are a family of 21Ϸ24-nt noncoding RNAs that regulate gene expression based on sequence similarity to their targets. Mammalian viruses including EBV, Kaposi's sarcoma-associated herpesvirus, human cytomegalovirus, and SV40 encode viral miRNAs (9). Viral encoded miRNAs were predicted for HSV-1 (10, 11) and also for HSV-2 (10). However, no miRNAs have been identified in HSV-2 or HSV-1 LAT sequences. Results HSV-2 LAT Exon 2 Encodes a miRNA.To find miRNAs within the HSV-2 LAT region, a plasmid containing the LAT sequence and its promoter (pSSK) and a mutant plasmid expressing LAT under control of the CMV-IE promoter (pCMV-SSK) were transfected into 293 cells. Small RNAs isolated from the transfected cells were cloned and sequenced. A 22-23-nt HSV-2 RNA sequence (designated HSV-2 miR-I) appeared at a frequenc...

High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.Human papillomavirus (HPV) infection of epidermal or mucosal epithelial cells causes benign and sometimes malignant neoplasms. Certain HPVs, such as HPV16, -18, -31, and -45, are detected frequently in anogenital cancers, particularly cancer of the cervix and anus, and are thus considered to be high risk or oncogenic (22,42). Among the high-risk HPVs, HPV16 and -18 cause Ͼ90% of cervical cancers (22) and Ͼ20% of oral cancers (35). High-risk HPVs encode two potent viral oncoproteins, E6 and E7, that mediate, respectively, degradation of the cellular proteins p53 and retinoblastoma protein (pRb), two tumor suppressor proteins that are essential for cell cycle control (21).In both HPV16 and HPV18, E6 and E7 are transcribed as a single bicistronic E6E7 transcript using a common promoter and a common early polyadenylation site (1). Promoter p97, upstream of the HPV16 E6 (16E6) open reading frame (ORF), and promoter p105, upstream of the HPV18 E6 (18E6) ORF, are responsible for the initiation of transcription in each virus genome. The bicistronic E6E7 pre-mRNAs of HPV16 and HPV18 contain three exons and two introns. One of the two introns, intron 1 (the cap-proximal intron), is positioned in the E6 ORF in both the HPV16 and HPV18 E6E7 pre-mRNAs ( Fig. 1A and B). Conceivably, intron 1 removal by RNA splicing would disrupt the E6 ORF and prevent full-length E6 from...

We recently identified an acutely and latently expressed viral microRNA (miRNA), miR-I, encoded by herpes simplex virus 2 (HSV-2) latency-associated transcript (LAT) through small RNA cloning and two miRNAs encoded by HSV-1 LAT through prediction. We now report the use of high-throughput sequencing technology to identify two additional relatively less-abundant viral miRNAs, miR-II and miR-III, encoded by HSV-2 LAT exon 2. miR-II includes two miRNAs, miR-II-5p and miR-II-3p, which are processed from the same miRNA precursor. miR-II and miR-III map antisense to the 5 untranslated region of ICP34.5 and to the coding region of ICP0 exon 3, respectively. These novel miRNAs are conserved in different HSV-2 strains, and their presence in infected-and transfected-cell cultures was confirmed by Northern hybridization. All three HSV-2 LAT-encoded miRNAs map to genome locations similar to those of three out of four identified HSV-1 LAT-encoded miRNAs, but the sequences of these miRNAs are not conserved. The expression of LAT-encoded miRNAs is negatively regulated by ICP4, the major viral transactivator. We further show that, similar to miR-I, miR-II is able to efficiently silence the expression of ICP34.5, a key viral neurovirulence factor, and that miR-III is able to silence the expression of ICP0, a key viral transactivator. All these data suggest that LAT sequences likely contribute to HSV latency and reactivation through tight control of these LAT-encoded miRNAs and their viral targets.Herpes simplex virus 1 (HSV-1) and HSV-2 are closely related herpesviruses. HSV-1 typically infects the facial region and establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia, while HSV-2 typically infects the genital region and establishes a lifelong latent infection in sensory neurons of the sacral dorsal root ganglia. Periodically, either virus may reactivate to cause symptomatic or asymptomatic recurrences in the area served by these sensory neurons. HSV-2 and HSV-1 have similar latent transcription patterns, in which the latency-associated transcript (LAT) is transcribed from within the genomic long repeats. In contrast to other viral promoters, the LAT promoter is highly active during latency, and LAT is the only viral gene product that is readily detectable during latency (39). HSV-1 LAT expression is inhibited by ICP4, the major viral transactivator required for most post-␣ gene expression (9, 12, 24), through an ICP4 binding site near the LAT transcription initiation site (14). The LAT introns (ϳ2.2 kb in HSV-2 and ϳ2 kb and 1.4 kb in HSV-1), which overlap the ICP0 transcript in an antisense direction, are much more abundant and stable than the ϳ8.5-kbp primary LAT transcript (13), which overlaps both the ICP0 and ICP34.5 transcripts in an antisense direction. ICP0 can transactivate a number of viral and host genes and is essential for HSV reactivation (4,5,18,19). ICP34.5, a key viral neurovirulence factor, is a protein kinase R inhibitor and is required for efficient viral replication in neurons...

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC. oncogenes E6 and E7 | noncoding RNAs | regulatory RNAs | virol oncogenesis | DNA tumor viruses

Bacillus subtilis var. natto N21 (Bac; for greater proteolytic capacity) and Saccharomyces cerevisiae Y10 (Sac; for greater acidic capacity) were applied to produce a 2-stage combined fermentation feed. This study investigated whether the enhancement of Bac+Sac fermented feed on broiler growth performance was due to the probiotics per se or due to the fermentation process. Trial 1 included 1-d-old broiler chicks (n=144) randomly assigned to control, water added (same as in the fermentation feed, 23%), and Bac+Sac fermented feed (FBac+Sac) treatments with 4 replicates. Trial 2 included 21-d-old broiler chickens (n=12) assigned into control and FBac+Sac groups for a metabolic trial for nutrient availability. Trial 3 included 1-d-old male broiler chicks (n=216) randomly assigned into 6 treatments with 3 replicates. Treatments included a control, Sac fermented feed (FSac), FBac+Sac, Bac powder (PBac), Sac powder (PSac), and Bac+Sac powder (PBac+Sac). The results from trial 1 showed that FBac+Sac increased BW and feed intake (P<0.05) in 21- and 39-d-old chickens. The water-added group showed decreased BW, weight gain, and feed intake (P<0.05). Trial 2 showed that FBac+ Sac increased gross energy availability (P<0.05). Trial 3 showed that FBac+Sac increased 21- and 39-d-old BW and weight gain (P<0.05). Diets supplemented with probiotic powder or fermented with Sac did not improve broiler growth performance (P>0.05). The growth performance improvement of the FBac+Sac treatment was probably not due to the added water, probiotic powder inclusion, or through single-strain fermentation, but due to the 2-stage fermentation process using Bac and Sac strains.

Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/ miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (U L ) region of the genome, 3 to the U L 15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

The herpes simplex virus (HSV) infected cell culture polypeptide 27 (ICP27) protein is essential for virus infection of cells. Recent studies suggested that ICP27 inhibits splicing in a gene-specific manner via an unknown mechanism. Here, RNA-sequencing revealed that ICP27 not only inhibits splicing of certain introns in <1% of cellular genes, but also can promote use of alternative 5′ splice sites. In addition, ICP27 induced expression of pre-mRNAs prematurely cleaved and polyadenylated from cryptic polyadenylation signals (PAS) located in intron 1 or 2 of ∼1% of cellular genes. These previously undescribed prematurely cleaved and polyadenylated pre-mRNAs, some of which contain novel ORFs, were typically intronless, <2 Kb in length, expressed early during viral infection, and efficiently exported to cytoplasm. Sequence analysis revealed that ICP27-targeted genes are GC-rich (as are HSV genes), contain cytosine-rich sequences near the 5′ splice site, and have suboptimal splice sites in the impacted intron, suggesting that a common mechanism is shared between ICP27-mediated alternative polyadenylation and splicing. Optimization of splice site sequences or mutation of nearby cytosines eliminated ICP27-mediated splicing inhibition, and introduction of C-rich sequences to an ICP27-insensitive splicing reporter conferred this phenotype, supporting the inference that specific gene sequences confer susceptibility to ICP27. Although HSV is the first virus and ICP27 is the first viral protein shown to activate cryptic PASs in introns, we suspect that other viruses and cellular genes also encode this function. (ICP27), an immediate early (IE) gene (among those first expressed after virus enters the cells) that is required for expression of some early and late viral genes as well as for virus growth, is highly conserved between HSV-1 and -2, two closely related neurotropic herpesviruses (1). ICP27 has a role in transcriptional regulation through association with the C-terminal domain of RNA polymerase II (2, 3), forms homodimers (4, 5), interacts with U1 small nuclear ribonucleoprotein (snRNP) through its C-terminal domain, and colocalizes with U1 and U2 snRNPs (6, 7). It also interacts with splicing factors such as SRSF3, SRSF1, SRSF7, and SRSF2 (8-11), and is involved in nuclear export of some viral transcripts (12, 13). The role of ICP27 in regulating pre-mRNA splicing remains controversial. Early studies indicated that, in an in vitro pre-mRNA splicing system, ICP27 may nonspecifically inhibit host pre-mRNA splicing, impairing spliceosome assembly as a result of interaction with SR protein kinase 1 (SRPK1) through ICP27's N-terminal RGG RNA-binding motif and/or interaction with spliceosome-association protein 145 (SAP145 or SF3B2) through ICP27's C-terminal domain (8, 11). A recent communication reported that HSV-1 does not inhibit cotranscriptional splicing and proposed that previous reports of ICP27-induced splicing inhibition were artifacts, due to misinterpretation of run-on transcription (14). Indeed, splicing of ...