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During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5′-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

Introduction/Background Epithelial ovarian cancer (EOC) is one of the deadliest gynaecologic malignancies worldwide. Nerve Growth Factor (NGF) and its high affinity receptor TRKA increase during epithelial ovarian cancer progression, promoting cell proliferation and angiogenesis, by increasing several oncogenic proteins. Many reports have showed that microRNA-145 (miR-145) is down-regulated in EOC, promoting the increase of oncoproteins. Our aims of were to evaluate whether NGF changes the miR-145 levels in EOC cells and the role of this miR in vitro and in vivo experiments. Methodology The human tissues and blood samples were obtained with informed consent and approved by Ethic Committee of our Institution. MicroRNA-145 levels were measured by qPCR in EOC samples, EOC cells lines (A2780 and SKOV3) and in serum samples. NGF action on miR-145 levels in EOC cell lines were evaluated using an inhibitor of TRKA (high affinity receptor of NGF). The over-expression of miR-145 in EOC cells were done by lipofectamine 2000 and by pGPGLenti-CMV-GFP vector. As control we also used the empty vector. After the transfection assay it was evaluated the migration and invasion of A2780 and SKOV3 cells. After stable miR over-expression assays, the GFP-expressing cells were sorted using GFP mark and the in vivo assays were done. Animal studies were conducted in accordance with appropriate guidelines of Ethical Committee of the Institution. Two groups of Immune-compromised NOD/SCID mice were injected with 8 x106 cells (intraperitoneal and subcutaneous) of A2780 or SKOV3 cells. Results NGF decreased the miR-145 levels in A2780 and SKOV3 cell lines. Also, when an inhibitor of TRKA receptor was used, levels of this miR increased. Beside, miR-145 levels decreased with the EOC progression in both tissues and plasma samples. MicroRNA-145 over-expression in EOC cells decreased cell proliferation and invasion. Also, the stable over-expression of miR-145 in EOC cells lines decreased malignant ascites presence in the animals. On the other hand, a delay in the tumour formation was observed in EOC cells which over-express miR 145 in subcutaneous lateral flank compared with the mice injected with control EOC cells. Conclusion These results suggest that overexpression/delivery of miR-145 could be a future adjuvant therapy in epithelial ovarian cancer Disclosures The authors declare that there is no conflict of interest. This work was funded by Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) N° 1160139 to CR

Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt-related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR-34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin (OPN/SPP1). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next-generation RNA-sequencing, protein-based assays, as well as the loss- and gain-of-function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell-cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

Resumen A 46 años de la identificación de los primeros polyomavirus en humanos (PyV), la preocupación por encontrar nuevos tipos relacionados a patologías de distintos órganos en pacientes inmunosuprimidos persiste. Hasta el momento de esta revisión, 15 PyV han sido descritos, muchos de ellos sin estar claramente asociados a enfermedades. En nuestro país, al igual que en gran parte de Sudamérica, el conocimiento y la pesquisa de estos agentes infecciosos son insuficientes por lo que sistematizamos aquello que se sabe sobre estos virus y su relación con los diferentes sistemas del cuerpo humano, con énfasis en los inmunosuprimidos y señalamos aquellos datos publicados en nuestro continente. Esperamos así incentivar un mayor estudio de estas infecciones virales.

Introduction In many type of cancer, including epithelial ovarian cancer (EOC), nerve growth factor (NGF) and its high affinity receptor TRKA are over-expressed and also are involved in proliferation and angiogenesis. On the other hand, many inflammatory molecules such as cyclooxigenase-2 (COX-2) and prostaglandins (PGE2) are over-expressed in cancer and also involved in the progression of cancer. Besides, a deregulation of some miRs expression has been described in EOC including miR-145 among others. The relationship of NGF-mirRs-COX2 in EOC has not been studied. Then, the aim of this work was to evaluate COX-2 and miR-145 levels in EOC progression and the changes of COX-2 and miR-145 levels by NGF action in epithelial ovarian cancer cell line (A2780). Also we evaluated COX-2 levels by Western-Blot after over-expression of miR-145 in A2780 cell line. Material and methods Thirty human ovarian tissues (normal, tumour and cancer) were obtained from Obstetrics and Gynaecology Department, Clinical Hospital, University of Chile and also from National Institute of Cancer with written consent and authorised by Ethic Committee of the Institutions. The COX-2 levels in tissues and cells were measured by RT-PCR, IHQ, and Western-Blot and miR-145 was measured by qPCR. Cells A2780 were stimulated with NGF (100 and 150 ng/ml) to measure COX-2 and miR-145 levels. Besides, over-expression of miR-145 was done by transfection assays with lipofectamine-2000 to evaluate cellular invasion and also transduction assays with lenti-virus in order to evaluate COX-2 levels by Western-Blot in A2780 cell line. Results and discussions It was found a significant decrease of miR-145 levels in EOC tissues compared with tumour tissues and normal ovarian tissues (p Conclusion MicroRNA-145 acts as tumour suppressor microRNA in ovarian cancer and these results suggest that miR-145 could be a good therapy for EOC. This work was supported by Grant: Fondecyt 1160139 to CR and ACCDIS - Conicyt-Fondap 15130011

Genomic replication and partial assembly of Rotavirus takes place in cytoplasmic viral structures called viroplasms. NSP5 is a viral phosphoprotein localized in viroplasms and its expression is imperative for viral cycle progress. During infection three isoforms of NSP5 can be observed by SDS-PAGE (26, 28 and 33–35 kDa) and previous reports suggested that they differ in their phosphorylation patterns. In this study we obtained NSP5 from infected cells and by mass spectrometry we were able to identify nine phosphorylation sites. We detected that in all the isoforms the same residues can be found either phosphorylated or unmodified. Quantitative analysis showed that the 28 kDa isoform has a higher phosphorylation level than the 26 kDa isoform suggesting that migration properties depend on the total number of phosphorylated residues. Moreover, we identified two not previously described modifications for this protein: an N-acetylation in Serine-2 and an intramolecular disulfide bond in a highly conserved motif, CXXC which is located between two charged alpha-helix motifs.

Polyomavirus BK (BKPyV) and JC (JCPyV) are persistent pathogens able to reactivate in im-munocompromised patients, involving mostly urinary and central nervous system. There are no Chilean studies in HIV positive patients.To detect BKPyV and JCPyV in blood of Chilean HIV positive adult patients and to correlate these results with clinical-related variables.96 stored blood samples from HIV patients belonging to the north area of Santiago were analyzed. Viral genomes of both viruses were detected by real-time PCR. For statistical analysis, chi-square (Pearson) and Mann-Whitney tests were used and p-values0.05 were considered significant.33% of the samples were positive for BKPyV and a significant correlation was found between the presence of BKPyV genome and the absence of detectable HIV viral load. We demonstrated the need to consider more than one amplification target to detect the BKPyV genome. All the samples were negative for JCPyV genome.BKPyV prevalence in Chilean HIV patients is higher than most of international studies. New studies regarding the interaction between both viruses are required. These patients should undergo periodic evaluations by urologist and nephrologist.

To identify clinical parameters in association with human papilloma virus (HPV) genotypes and histopathology diagnosis in HIV-positive patients with external condylomata acuminata (ECA), 400 Chilean HIV-positive patients were included in the study. Forty-seven patients presented ECA. Clinical parameters and socio demographic data were recorded. Histopathology study and HPV linear array genotyping assay were performed. Intraepithelial neoplasia (IEN) grade 2 or 3 was found in 8.5% of patients, associated to HPV-16. Patients were mainly single, MSM, with history of sexually transmitted disease (STD), multiple sexual partners, receiving antiretroviral therapy and with recurrent lesions. All ECA were mainly perianal, grey or pink colored, exophytic with less than two years evolution. No clinical parameter could predict the development of high grade IEN in HIV patients with ECA. It seems necessary to perform biopsy and genotype all HIV positive patients with ECA.

Human metapneumovirus (hMPV) is a significant cause of acute lower respiratory tract infection in all age groups, particularly in children. Two genetic groups and four subgroups of hMPV have been described. They co-circulate during an epidemic in variable proportions. The aims were to characterize the genotypes of hMPV recovered from children hospitalized for acute lower respiratory tract infection and to establish the molecular epidemiology of strains circulating in Santiago of Chile during a 2-year period. The detection of the N gene by reverse-transcription polymerase chain reaction was carried out for screening 545 infants hospitalized for acute lower respiratory tract infection in Santiago during 2003‐2004. The genetic typing of hMPV was performed by analyzing the fusion gene sequences. hMPV was detected in 10.2% (56/545 cases). Phylogenetic analysis of F gene sequences from 39 Chilean hMPV strains identified the two groups and four subgroups previously described. Strains clustered into group A were split further into the sub lineages A1, A2, and A3. Most Chilean strains clustered into the proposed novel A3 sub lineage (59%). A3 viruses were present in both years, while A1 and A2 circulated just in 1 year. In conclusion, hMPV is a relevant cause of acute lower respiratory infection in Chilean children and the potential novel cluster of group A emphasize the need for further regional genetic variability studies. J. Med. Virol. 81:340–344

We have previously shown a functional interaction between human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins and cigarette smoke condensate (CSC) in lung cells suggesting cooperation during carcinogenesis. The molecular mechanisms of such interaction, however, remain to be elucidated. Here we first present evidence showing that cigarette smoke condensate (CSC) has the ability to activate the HPV-16 p97 promoter by acting on the long control region (LCR) in lung epithelial cells. Interestingly, we observed that CSC-induced p97 promoter activation occurs in a dose-dependent manner in both tumor A-549 (lung adenocarcinoma), H-2170 (bronchial carcinoma), SiHa or Hela (cervical carcinoma) cells but not in non-tumor BEAS-2B (bronchial) or NL-20 (alveolar) lung cells unless they ectopically expressed the HPV-16 E6 and E7 oncogenes. In addition, we also observed a significant increase of primary DNA damage in tumor and non-tumor CSC-treated lung cells expressing HPV-16 E6 and E7 oncogenes suggesting a cooperative effect in this process, even though the contribution of E7 was significantly higher. Taken together, our results strongly suggest that tobacco smoke is able to induce the activation of the HPV-16 p97 promoter in cooperation with HPV-16 E6 and E7 oncogenes that, in turn, sensitize lung cells to tobacco smoke-induced DNA damage.

Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.