Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPVnegative cancers arising in the same tissue.

Using highly sensitive microarray-based procedures, we identified eight microRNAs (miRNAs) showing robust differential expression between 31 laser-capture-microdissected nasopharyngeal carcinomas (NPCs) and 10 normal healthy nasopharyngeal epithelial samples. In particular, miRNA mir-29c was expressed at one-fifth the levels in tumors as in normal epithelium. In NPC tumors, the lower mir-29c levels correlated with higher levels of multiple mRNAs whose 3 UTRs can bind mir-29c at target sequences conserved across many vertebrates. In cultured cells, introduction of mir-29c down-regulated these genes at the level of mRNA and inhibited expression of luciferase encoded by vectors having the 3 UTRs of these genes. Moreover, for each of several genes tested, mutating the mir-29c target sites in the 3 UTR abrogated mir-29c-induced inhibition of luciferase expression. Most of the mir-29c-targeted genes identified encode extracellular matrix proteins, including multiple collagens and laminin ␥1, that are associated with tumor cell invasiveness and metastatic potential, prominent characteristics of NPC. Thus, we identify eight miRNAs differentially expressed in NPC and demonstrate the involvement of one in regulating genes involved in metastasis.microarray ͉ collagen ͉ metastasis ͉ miRNA M icroRNAs (miRNAs) are short (Ϸ22 nucleotides) noncoding RNAs involved in posttranscriptional silencing of target genes. In animals, miRNAs control expression of target genes by inhibiting translation, by degrading target mRNAs, or both, through binding to their 3Ј UTRs with varying degrees of sequence complementarity (1). miRNAs have been found to regulate genes involved in diverse biological functions, including development, differentiation, proliferation, and stress response (2). Recently, a growing number of miRNAs have been implicated in cancers, including mir-15 and mir-16 in B cell chronic lymphocytic leukemias (3, 4); mir-143 and mir-145 in colorectal cancer (5); mir- 17-5p, mir-21, mir-125b, mir-145, and mir-155 in breast cancer (6, 7); mir-19, mir-146, mir-181b, mir-221, mir-222, and mir-346 in thyroid cancer (8-10); and mir-21 in glioblastoma (11). A significant number of miRNAs also have been mapped to cancer-associated genomic regions (12). Expression of miRNA let-7 has been correlated with prognosis in lung cancer (13) and found to regulate Ras in the same tumor (14). Very recently, mir-10b has been shown to contribute to metastasis in breast cancer (15). Although many miRNAs have been implicated in regulating cancers, very few of their target genes, and hence their downstream mode of action, have been identified.We developed a sensitive microarray-based assay to profile miRNA expression and used it to analyze human miRNAs in laser-microdissected tumor and normal cells from biopsies of a highly invasive cancer, nasopharyngeal carcinoma (NPC), and site-matched normal tissues. Eight miRNAs were differentially expressed. One of them, mir-29c, down-regulated in NPC, was shown to target multiple mRNAs encoding extracellular ma...

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona-and toroviruses. The gene contains two large ORFs, ORFla and ORFlb, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORFlb is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORFlb product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORFlb products. The sequences of the EAV and coronavirus ORFla proteins were found to be much more diverged. The EAV ORFla product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

Positive-strand RNA virus genome replication is invariably associated with extensively rearranged intracellular membranes. Recent biochemical and electron microscopy analyses, including three-dimensional electron microscope tomographic imaging, have fundamentally advanced our understanding of the ultrastructure and function of organelle-like RNA replication factories. Notably, for a range of positive-strand RNA viruses embodying many major differences, independent studies have revealed multiple common principles. These principles include that RNA replication often occurs inside numerous virus-induced vesicles invaginated or otherwise elaborated from a continuous, often endoplasmic reticulum-derived membrane network. Where analyzed, each such vesicle typically contains only one or a few genome replication intermediates in conjunction with many copies of viral nonstructural proteins. In addition, these genome replication compartments often are closely associated with sites of virion assembly and budding. Our understanding of these complexes is growing, providing substantial new insights into the organization, coordination, and potential control of crucial processes in virus replication.

Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses.

Equine arteritis virus (EAV) is a positivestrand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

A prespecified set of genes may be enriched, to varying degrees, for genes that have altered expression levels relative to two or more states of a cell. Knowing the enrichment of gene sets defined by functional categories, such as gene ontology (GO) annotations, is valuable for analyzing the biological signals in microarray expression data. A common approach to measuring enrichment is by crossclassifying genes according to membership in a functional category and membership on a selected list of significantly altered genes. A small Fisher's exact test p-value, for example, in this 2 × 2 table is indicative of enrichment. Other category analysis methods retain the quantitative gene-level scores and measure significance by referring a category-level statistic to a permutation distribution associated with the original differential expression problem. We describe a class of random-set scoring methods that measure distinct components of the enrichment signal. The class includes Fisher's test based on selected genes and also tests that average gene-level evidence across the category. Averaging and selection methods are compared empirically using Affymetrix data on expression in nasopharyngeal cancer tissue, and theoretically using a location model of differential expression. We find that each method has a domain of superiority in the state space of enrichment problems, and that both methods have benefits in practice. Our analysis also addresses two problems related to multiple-category inference, namely, that equally enriched categories are not detected with equal probability if they are of different sizes,

To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1-CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/ CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA− precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1−, CD34−, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma-tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.cervical cancer | HPV | estrogen | tumor microenvironment | stroma G lobally, cervical cancer (CxCa) is the second most common cancer in women, with >500,000 new cases each year, half of which are ultimately fatal (1). In the developed world, routine CxCa screening for abnormal cervical cytology, human papillomavirus (HPV), or both (2) has strongly reduced CxCa incidence, demonstrating the value of recognizing and removing early neoplasms (3). Such screening programs present a rare opportunity to study the sequential molecular changes in the development of a human cancer (4).CxCa development is related to infection with high-risk oncogenic HPVs, most prominently HPV16 and HPV18 (5-7). HPV oncoproteins E6 and E7 are best known for blocking tumor suppressor functions of p53 and Rb, respectively, but have numerous additional interaction partners (8-13). Most cervical HPV infections are cleared (14), but in a fraction of cases persistent infections lead to increasingly severe grades of dysplasia (cervical intraepithelial neoplasia grades 1, 2, and 3; CIN1, CIN2, CIN3) and ultimately to invasive cancer. Although the key steps of the carcinogenic process, HPV infection, progression to precancer, and invas...