A mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral MicroRNAs.

Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA polymerase II and then processed by the Drosha endonuclease to generate approximately 60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer to generate mature miRNAs. Previously, some short introns, called miRtrons, were reported to fold into pre-miRNA hairpins after splicing and debranching, and miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin RNAs. Here we report that the miRNAs encoded by murine gamma-herpesvirus 68 (MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs are transcribed from RNA polymerase III promoters located within adjacent viral tRNA-like sequences. The resultant pri-miRNAs, which bear a 5' tRNA moiety, are not processed by Drosha but instead by cellular tRNase Z, which cleaves 3' to the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to yield the mature viral miRNAs.

Cloning and analysis of microRNAs encoded by the primate gamma-herpesvirus rhesus monkey rhadinovirus.

Several pathogenic human herpesviruses have recently been shown to express virally encoded microRNAs in infected cells. Although the function of these microRNAs is largely unknown, they are hypothesized to play a role in mediating viral replication by downregulating cellular mRNAs encoding antiviral factors. Here, we report the cloning and analysis of microRNAs encoded by Rhesus Monkey Rhadinovirus (RRV), an animal virus model for the pathogenic human gamma-herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV). RRV expresses several microRNAs that are encoded in the same genomic location as the previously reported KSHV microRNAs, yet these microRNAs are unrelated in primary sequence. These data set the stage for the mutational ablation and phenotypic analysis of RRV mutants lacking one or more viral microRNAs.

The imprinted H19 noncoding RNA is a primary microRNA precursor.

Although H19 was the first imprinted noncoding transcript to be identified, the function of this conserved RNA has remained unclear. Here, we identify a 23-nucleotide microRNA derived from H19 that is endogenously expressed in human keratinocytes and neonatal mice and overexpressed in cells transfected with human or mouse H19 expression plasmids. These data demonstrate that H19 can function as a primary microRNA precursor and suggest that H19 expression results in the post-transcriptional downregulation of specific mRNAs during vertebrate development.

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

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.

A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing.

MicroRNAs (miRNAs) are a class of approximately 22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3' untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed.

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

Transcriptional origin of Kaposi's sarcoma-associated herpesvirus microRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Use of RNA polymerase II to transcribe artificial microRNAs.

MicroRNAs (miRNAs) are endogenously encoded approximately 22-nt-long RNAs that are generally expressed in a highly tissue- or developmental-stage-specific fashion and that posttranscriptionally regulate target genes. Regulatable RNA polymerase II promoters can be used to overexpress authentic microRNAs in cell culture. Furthermore, one can also design and express artificial microRNAs based on the features of existing microRNA genes, such as the gene encoding the human miR-30 microRNA. Overexpression or inappropriate expression of authentic microRNAs may facilitate the study of their normal functions and expression of artificial microRNAs may permit effective, regulated RNA interference in vivo.

Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.

The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.

Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury.

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-gamma-inducible protein 10 (IP-10), Mx1, and guanylate-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.

Increased IFN-gamma protein in bronchoalveolar lavage fluid of anti-IP-10 antibody-treated F344 rats following Sendai viral infection.

A previous study showed that virus-resistant F344 rats had higher levels of interferon-gamma (IFN-gamma)-inducible protein 10 (IP-10) than did virus-susceptible BN rats early after Sendai viral infection. The initial goal of this study was to determine if an early high expression of IP-10 in F344 rats contributes to their resistance to virus-induced airway injury. Infected F344 rats were treated with anti-IP-10 rabbit serum or normal rabbit serum. Results indicated that blocking of IP-10 protein did not significantly change the resistance of F344 rats. However, we observed that neutralization of IP-10 increased IFN-gamma protein in bronchoalveolar lavage (BAL) fluid of F344 rats 7 days after inoculation compared with rats that received normal rabbit serum. The pulmonary IFN-gamma mRNA abundance remained comparable. This effect was not caused by fluctuation of the viral titer in the lung. This interesting phenomenon suggests that expression of IFN-gamma protein can be modulated by treatment with anti-IP-10 antibody at the posttranscriptional or translational level in this model.

Cloning of Schistosoma japonicum Chinese Strain TPI Gene and Characterization of Its Expression Product in Escherichia coli.

Triose-phosphate isomerase is an important candidate for schistosoma antigens. An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA. Sequence analysis revealed that this fragment contained S. japonicum (Chinese strain) triose-phosphate isomerase gene. Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E. coli. The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography. Its molecular weight was determined to be 54 kD. The yield of expression was around 30 mg/L E. coli culture. Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S. japonicum multi-valent recombinant vaccine.

Cloning of Schistosoma japonicum Chinese Strain Fatty Acid Binding Protein (Sj-FABPc) Gene and Its Overproduction in Escherichia coli.

A 600 bp DNA fragment was amplified by PCR, from an adult Schistosoma japonicum cDNA library. Sequence analysis revealed that this fragment containedthe S. japonicum Chinese Mainland strain fatty acid binding protein (Sj-14FABPc) gene. This gene was then cloned into the expression vector pGEX-2T, and subsequently expressed in Escherichia coli. The recombinant GST-fusion protein could be purified by glutathione agarose affinity chromatography. Its molecular weight was about 41 kD. The yield of expression was around 25 mg/L E. coli culture. The immunological test suggested that the recombinant protein had good antigenicity, and could be developed into a new vaccine molecule of S. japonicum.