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The consequences of Zika virus (ZIKV) infections were limited to sporadic mild diseases until almost a decade ago, when epidemics outbreaks took place and were followed by virus introduction and quick spread into the Americas. Simultaneously, novel severe neurological manifestations for ZIKV infections were identified, including congenital microcephaly. However, the complete understanding of why the epidemic strains behave differently from previously circulating variants is still missed and many questions still remain about the actual significance of ZIKV genetic variations in the epidemiology and biology of ZIKV. Here, we attained to analyze a high number of sequences to identify genes with different levels of variability and patterns of genomic variations that could be associated with ZIKV diversity. We compared numerous epidemic strains with the pre-epidemic ones from both the Asian and the African lineages, while we also investigated special variations among the epidemic ZIKV strains derived from microcephaly cases. We identified several viral genes with dissimilar mutation rates among the ZIKV strain groups. Moreover, we found novel protein variation profiles that might be associated with the epidemiological particularities of each category of ZIKV strains. Finally, we assessed the impact of the detected changes on the structure and stability of the corresponding viral proteins, finding some interesting non synonymous variations which could help to explain the heterogeneous features among the diverse ZIKA strains. Altogether, this work contributes to identify ZIKV genetic variations that might have a phenotypical impact, as a basis for future experimental analysis to elucidate the recent ZIKV emergency.

Hepatitis E virus (HEV) is a public health concern due to its zoonotic transmission to human, being pigs a highly recognized reservoir. We previously demonstrated HEV genotype 3 infections in pig herds from the highest commercial active region from Argentina. Here, we present a case of acute symptomatic hepatitis E in an elderly man with occupational exposure to pigs who referred regular consumption of pork and sausages. HEV infection in this patient was demonstrated by serological methods, as well as by HEV RNA detection in serum and stool samples using the HEV/MS2 duplex RT-qPCR, formerly optimized in our laboratory. We further detected HEV RNA in pig faeces from the patient´s farm. To confirm the potential role of swine in the transmission, we performed a phylogenetic analysis of all HEV RNA derived from both, the patient and the pig samples. A 303 nt region within the HEV 5 'ORF2 was amplified by nested RT-PCR and subsequently sequenced. Phylogenetic analysis showed that the strains isolated from the farmer and from his pigs presented a nucleotide identity of 100%. These results support the zoonotic transmission of circulating HEV strains and confirm this epidemiological association for the first time in Argentina.

Human papillomavirus (HPV) infection is a multistep process that implies complex interactions of the viral particles with cellular proteins. The HPV capsid includes the two structural proteins L1 and L2, which play crucial roles in infectious viral entry. L2 is particularly relevant for the intracellular trafficking of the viral DNA toward the nucleus. Here, using proteomic studies we identified CCT proteins as novel interaction partners of HPV-16 L2. The CCT multimeric complex is an essential chaperonin which interacts with a large number of protein targets. We analyzed the binding of different components of the CCT complex to L2. We confirmed the interaction of this structural viral protein with the CCT subunit 3 (CCT3), and we found that this interaction requires the N-terminal region of L2. Defects in HPV-16 pseudoviral particle (PsV) infection were revealed by small interfering RNA-mediated knockdown of some CCT subunits. While a substantial drop in the viral infection was associated with the ablation of CCT component 2, even more pronounced effects on infectivity were observed upon depletion of CCT component 3. Using confocal immunofluorescence assays, CCT3 colocalized with HPV PsVs at early times after infection, with L2 being required for this to occur. Further analysis showed the colocalization of several other subunits of CCT with the PsVs. Moreover, we observed a defect in capsid uncoating and a change in PsV intracellular normal processing when ablating CCT3. Taken together, these studies demonstrate the importance of CCT chaperonin during HPV infectious entry. IMPORTANCE Several of the mechanisms that function during the infection of target cells by HPV particles have been previously described. However, many aspects of this process remain unknown. In particular, the role of cellular proteins functioning as molecular chaperones during HPV infections has been only partially investigated. To the best of our knowledge, we describe here for the first time a requirement of the CCT chaperonin for HPV infection. The role of this cellular complex seems to be determined by the binding of its component 3 to the viral structural protein L2. However, CCT’s effect on HPV infection most probably comprises the whole chaperonin complex. Altogether, these studies define an important role for the CCT chaperonin in the processing and intracellular trafficking of HPV particles and in subsequent viral infectious entry.

Hepatitis E virus (HEV) is currently considered as a global health concern due to the recognition of its zoonotic transmission to humans, mainly from swine, and its association with the development of severe cases of hepatitis in human risk populations. The lack of updated data on HEV state of infection in swineherds of Argentina, and the necessity of robust technologies for its detection in complex biological samples, positions HEV as an emerging issue in public health. Here, we have optimized a RT-qPCR with internal control for a more precise and accurate HEV RNA detection in swine stool samples. We implemented this optimized molecular tool to analyse the current epidemiological scenario of HEV infection in swine from the core region of commercial activity of Argentina. A total of 135 stool samples were collected from 16 different farms and tested for HEV presence, resulting in 11 positive cases (8.1%). Phylogenetic analysis demonstrated that all of them correspond to HEV genotype 3 and that different subtypes circulate in the region. Moreover, two of the detected strains presented a high nucleotide similarity with a previously identified isolate from human sewage discharges, suggesting the zoonotic transmission of HEV to humans. Collectively, this work provides a better understanding of HEV epidemiology in Argentina while contributes to the improvement of HEV detection technologies.

Background Persistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions. Methods Due to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively. Results We demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation. Conclusions In general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.

Background: Epstein Barr Virus (EBV) infection has been associated with the Rheumatoid Arthritis (RA) etiopathology, thus, considering it a relevant etiological agent in the disease development. We have previously observed the Hyaluronic Acid (HA) serum increase in active RA patients (DAS 28-4 > 2.6) which might be responsible for the erythrocyte deformability, estimated by the erythrocyte Rigidity Index (RI). Objective: In the present study, we analyzed in one hundred RA patients if anti-EBV serum antibodies (TsEBV) and HA and RI levels are related with the disease activity. Results: [HA] s [[controls]]: 20.0 ± 9; [HA] s [[AR]]: 155.8 ± 44 (p< 0.00001 vs. controls (n:40). Antibodies anti-EBV (1/ TsEBV) [[controls]]: 2.55 ± 0.49; [[RA]]: 1.85 ± 0.35 (p< 0.00001 vs. controls). There were significant correlations between DAS 28-4 vs. log2 1/TsEBV (r: 0.70, p < 0.00001) and DAS 28-4 vs. RI (r: 0.75, p < 0.00001) in active RA patients. However, we observed a strong positive correlation between [HA] s in active RA patients vs. log2 1/TsEBV (r: 0.83, p < 0.00001) and [HA] s vs. RI (r: 0.91, p < 0.00001). Conclusion: The correlation obtained points out that the TsEBV may be related with the degree of RA activity determined by DAS28-4 and [HA]s in these patients. There was also a decrease in erythrocyte deformability estimated as RI, which is correlated with the DAS 28-4 activity index. Therefore, RI can be used as a reliable marker of RA activity. These findings might suggest a role of EBV infection regarding an autoimmune mechanism present in RA. Fil: Perez, Germán. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentina Fil: Svetaz, María José. Universidad Nacional de Rosario; Argentina Fil: Leda Urli. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina Fil: Volpintesta, Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina Fil: Taborda, Miguel Ángel. Universidad Nacional de Rosario; Argentina Fil: Gardiol, Daniela Nora. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentina Fil: Gir, Adriana. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentina Fil: Luquita, Alejandra Nora. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina. Universidad Nacional de Rosario; Argentina

Zika Virus (ZIKV) is an RNA virus that belongs to the Flavivirus (FV) genus. In the last years, several unique characteristics of ZIKV among FV have been revealed, as the multiple routes of transmission and its ability to reach different human tissues, including the central nervous system. Thus, one of the most intriguing features of ZIKV biology is its ability to cross diverse complex biological barriers. The main aim of this study is to contribute to the understanding of the still unclear mechanisms behind this viral activity. We investigated an African strain and two South American ZIKV isolates belonging to the Asian lineage, in order to characterize possible differences regarding their ability to disturb intercellular junctions. The Asian isolates correspond to an imported (Venezuelan) and an autochthonous (Argentinian) ZIKV strain for which there is still no data available. We focused on occludin and DLG1 expression as markers of tight and adherent junctions, respectively. For this, we applied a quantitative immunofluorescence assay that can ascertain alterations in the cell junction proteins expression in the infected cells. Our findings indicated that the different ZIKV strains were able to reduce the levels of both polarity proteins without altering their overall cell distribution. Moreover, the grade of this effect was strain-dependent, being the DLG1 reduction higher for the African and Asian Venezuelan isolates and, on the contrary, occludin down-regulation was more noticeable for the Argentinian strain. Interestingly, among both junction proteins the viral infection caused a relative larger reduction in DLG1 expression for all viruses, suggesting DLG1 may be of particular relevance for ZIKV infections. Taken together, this study contributes to the knowledge of the biological mechanisms involved in ZIKV cytopathogenesis, with a special focus on regional isolates.

Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain–containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions. Fil: Marziali, Federico Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bugnon Valdano, Marina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Avalos, Clarisse Brunet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moriena, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cavatorta, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Gardiol, Daniela Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Human Discs large tumour suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes. We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n = 30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n = 23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n = 4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n = 3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p

High-risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD-95/DLG/ZO-1 (PDZ) domain-containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ-dependent manner without significant reduction in Par3 protein levels. In addition, the high-risk HPV-18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV-associated tumours. Fil: Facciuto, Florencia Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bugnon Valdano, Marina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Marziali, Federico Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Massimi, Paola. International Centre For Genetic Engineering And Biotechnology; Italia Fil: Banks, Lawrence. International Centre For Genetic Engineering And Biotechnology; Italia Fil: Cavatorta, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Gardiol, Daniela Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

The general features of the PDZ domain structure and functions have been extensively studied during the last decade. PDZ domains are generally present in proteins that are involved in multiple interactions to assemble functional protein complexes that control key cellular processes. One of the best characterized functions of PDZ domain-containing proteins is control of epithelial cell polarity and cell-cell contacts. In the present review, we summarize the current knowledge on regulation of expression of certain PDZ polarity proteins localized at the intercellular junctions. In addition, we provide a critical overview of recent findings regarding the role of these proteins during development of human diseases. Complete understanding of these issues is valuable for the design of novel therapeutic intervention for common pathologies, such as cancer.

Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions. Fil: Bugnon Valdano, Marina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cavatorta, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Morale, M. G.. Universidade de Sao Paulo; Brasil Fil: Marziali, Federico Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Lino, V. de Souza. Universidade de Sao Paulo; Brasil Fil: Steenbergen, R. D. M.. VU University Medical Center; Países Bajos Fil: Boccardo, E.. Universidade de Sao Paulo; Brasil Fil: Gardiol, Daniela Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Human Disc large (DLG1) has been demonstrated to be involved in the control of cell polarity and maintenance of tissue architecture, and is frequently lost in human tumours. However, the mechanisms controlling DLG1 expression are poorly understood. To further examine the regulation of DLG1 expression, we analysed the 5' ends of DLG1 transcripts by rapid amplification of cDNA ends polymerase chain reaction. We identified an alternative splicing event in the 5' region of DLG1 mRNA that generates transcripts with two different 5' untranslated regions (5'-UTRs). We show by reporter assays that the DLG1 5'-UTR containing an alternatively spliced exon interferes with the translation of a downstream open reading frame (ORF). However, no significant differences in mRNA stability among the DLG1 5'-UTR variants were observed. Sequence analysis of the additional exon present in the larger DLG1 5'-UTR showed the presence of an upstream short ORF which is lost in the short version of the 5'-UTR DLG1. By mutagenesis and luciferase assays, we analysed the contribution of this upstream short ORF in reducing translation efficiency, and showed that its disruption can revert, to some extent, the negative regulation of large 5'-UTR. Using computational modelling we also show that the large DLG1 5'-UTR isoform forms a more stable structure than the short version, and this may contribute to its ability to repress translation. This represents the first analysis of the 5' region of the DLG1 transcripts and shows that differential expression of alternatively spliced 5'-UTRs with different translational properties could result in changes in DLG1 abundance.

A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways.

Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO 4 ·7H 2 O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings.

Human discs large (DLG1) has been demonstrated to be involved in cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 expression are not fully understood. This is relevant as DLG1 is lost during the later stages of malignant progression. We initiated a series of studies to analyse the mechanisms regulating DLG1 expression. We have previously reported the identification of an alternative splicing event in the 5 ′ untranslated region (5 ′ -UTR) of DLG1 mRNA that generates transcripts with two different 5 ′ -UTR (short and large 5 ′ -UTR variants). In this study, we further examined the impact of the DLG1 transcription and the role of the differential expression of the alternative 5 ′ -UTRs on DLG1 protein levels. We analysed these mechanisms during cell processes like differentiation, cell cycle progression and cell-cell contact formation, where the importance of DLG1 activities was previously established. The data presented in this report suggest that the transcriptional regulation of DLG1 strongly contributes to DLG1 abundance and that differential expression of alternative 5 ′ -UTRs with different translational properties, also cooperates, depending on the cell type and cell situation. This study provides new evidence for understanding the transcriptional regulation of DLG1 and the changes in DLG1 expression during different biological processes. Fil: Marziali, Federico Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cavatorta, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bugnon Valdano, Marina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Facciuto, Florencia Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Gardiol, Daniela Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Drosophila discs large (Dlg) has been shown to be an essential regulator of cell polarity and attachment, and is classified as a potential tumour suppressor in higher eukaryotes. Human Dlg is expressed in epithelial cells at sites of cell-cell contact and acts as a negative regulator of cell growth. Although hDlg has been shown to be phosphorylated during mitosis, little is known about its activity during this stage of the cell cycle. To investigate this further we have analysed in detail the pattern of hDlg expression during mitotic cell division. In early mitosis there is a marked increase in membrane-bound hDlg which is then retained throughout mitosis, while during cytokinesis, there is a specific concentration of hDlg at the midbody. Using mutants of Dlg we show that this is mediated by sequences in the carboxy terminal region of Dlg, but it does not require the SH3 or PDZ domains, and is independent of binding to protein 4.1. Finally, using a mutant of Dlg that consists of just this carboxy terminal region of the protein, we show that it can compete with endogenous hDlg for midbody accumulation, and this mutant also gives rise to altered cell growth. We conclude that localisation of Dlg to the midbody indicates a role for Dlg at this critical point in cytokinesis.

Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further defined the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal effect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the first 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.

The general features of the PDZ domain structure and functions have been extensively studied during the last decade. PDZ domains are generally present in proteins that are involved in multiple interactions to assemble functional protein complexes that control key cellular processes. One of the best characterized functions of PDZ domain-containing proteins is control of epithelial cell polarity and cell-cell contacts. In the present review, we summarize the current knowledge on regulation of expression of certain PDZ polarity proteins localized at the intercellular junctions. In addition, we provide a critical overview of recent findings regarding the role of these proteins during development of human diseases. Complete understanding of these issues is valuable for the design of novel therapeutic intervention for common pathologies, such as cancer.

PDZ domains are very abundant protein interaction domains widespread in nature. A large amount of evidence has underscored the importance of the PDZ interactions in the control of intracellular pathways whose abnormal regulation may lead to the development of several pathologies. This series of minireviews covers different aspects of human PDZ-containing proteins, underlining and discussing new concepts and findings.

The regulation of human papillomavirus (HPV) gene expression by the E2 protein is a critical feature of the viral life cycle. Previous studies have shown an important role in transcription for the ubiquitin-proteasome pathway, but its role in HPV gene expression has not been addressed. We now show that HPV E2 requires an active proteasome for its optimal transcriptional activator function. This involves an interaction with the Mdm2 ubiquitin ligase, which together with E2 acts synergistically to activate the HPV type 16 promoter. We also show that HPV E2 recruits Mdm2 onto HPV promoter sequences, providing an explanation for this cooperative activity.

A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

In developing countries, the introduction of human papillomaviruses (HPV) DNA testing as an adjunct to cytological screening programs has been delayed due to the lack of high performance and cost effective diagnostic nucleic acid methods. In this study we report the development and evaluation of the L1HPVPCR, a PCR-based method for the detection and typing of five of the most prevalent high-risk HPV types. The L1HPVPCR assay combines amplification with the MY09/11 HPV consensus primer system, liquid hybridization of the PCR products with no radioactive probes and enzyme immunoassay analysis. The technique is a user-friendly system that allows accurate HPV DNA detection and typing with inexpensive instrumentation that could be performed with not sophisticated reagents in almost any laboratory. Different cutoff points for generic and specific HPV detection were determined using reproducibility analysis and receiver operating characteristic curves to ensure good analytical sensitivity and clinical effectiveness. We used the L1HPVPCR assay to estimate the prevalence of HPV infection in 127 women at risk of cervical cancer from the city of Rosario (Argentina), where no epidemiological data has been previously reported. Further, we explored the clinical utility of the L1HPVPCR assay respect the Pap smear using a combined diagnosis of cytology, histology and colposcopy as gold standard. In conclusion, our results indicate that the assay described here provides a tool for accurate HPV DNA testing and could be applied in regions where no commercial tests are available.

Loss of cell polarity is one of the hallmarks of malignant carcinomas. Most of the understanding about the link between cell polarity and proliferation control comes from studies on the Drosophila tumor suppressors discs large (Dlg), scribble (Scrib) and lethal giant larvae (lgl). Mammalian homologues of these proteins have been described and are conserved in sequence and function. Human Dlg (hDlg) and Scrib were independently shown to be down-regulated during malignant progression. This, and other lines of evidence, points toward the participation of both hDlg and hScrib in a common pathway involved in polarity control and tumor suppression. We investigated the correlation between the expression of both proteins in tissues and their relative contributions to the maintenance of tissue architecture during colon cancer development. We analyzed the levels and distribution of hDlg and hScrib by immunohistochemistry, using serial sections of the same sample. We used normal and neoplastic colon mucosa, since it offers a good model for analyzing these features in progressive dysplastic stages. The results demonstrate that both proteins localize at the same regions in polarized colon epithelia, and that in normal samples the proteins' distribution varies as cells differentiate at the surface mucosa. In neoplasia, alterations in the expression pattern of hDlg and of hScrib increase during tumor progression; down-regulation of both proteins being associated with lack of epithelial cell polarity and disorganized tissue architecture. The results, therefore, demonstrate that there is an inverse relationship between the levels of hDlg and hScrib expression and the loss of cell polarity and tissue architecture in the colon. © 2006 Wiley-Liss, Inc.

High-risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h-Dlg for ubiquitin-mediated proteolysis. The h-Dlg oncosuppressor is associated with cell-cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h-Dlg expression in HPV-associated lesions. We analyzed h-Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR-colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPV-associated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV-positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late-stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability.

The discs large (Dlg) tumour suppressor protein is targeted for ubiquitin-mediated degradation by the high-risk human papillomavirus E6 proteins. To understand further the mechanisms behind this, a mutational analysis of Dlg was undertaken. This study demonstrates that an intact PDZ domain 2 (PDZ2) on Dlg is necessary for the ability of E6 to bind and degrade Dlg. However, additional residues within the amino-terminal portion of Dlg are also required for optimal E6 activity. Stable cell lines expressing different Dlg mutants were also established and these confirm that Dlg is regulated intrinsically by the proteasome in the absence of E6; however, in this case, the sequences responsible for regulating Dlg stability lie predominantly within PDZ2. These results suggest that there are at least two mechanisms for regulating Dlg protein stability and that the pathways used by E6 are not necessarily the same as those used in the cell in its absence.

The protein Kinase A (PKA) pathway was found to selectively regulate the function of oncogenic but not non-oncogenic E6 proteins. High risk E6 proteins are phosphorylated at their Dlg/PDZ binding motif at the C-terminus by a PKA like activity. This PKA and PDZ binding module is found only for human PV, is strictly conserved in all the transforming HPVs and is absent in all the low risk HPV types. We present evidence of a conditional regulation of E6 induced degradation of Dlg. HPV18E6 positive but not HPV negative keratinocytes exhibit increased Dlg steady state levels under conditions of high PKA activity, with a concomitant increase in the presence of Dlg at tight junctions. In vitro binding experiments show that E6 phosphorylation by PKA reduces its binding to Dlg and molecular modelling can explain this observation in a structural context. E6 dependent degradation of Dlg in cells with high PKA levels is inhibited and this is dependent on phosphorylation of the PDZ binding site in E6. In contrast, the degradation of p53 induced by E6 is not affected by PKA. We propose a differential regulation of E6 for the ubiquitin mediated degradation of specific E6 target proteins.

The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We first show that the differences in the affinities of HPV-16 and of HPV-18 E6 proteins for binding DLG is reflected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to define regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Significant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most significantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway.

An important characteristic of the E6 proteins derived from oncogenic associated human papillomaviruses (HPVs) is their ability to target the cellular tumour suppressor protein, p53, for ubiquitin mediated degradation. Several studies have attempted to address the important characteristics of both E6 and p53 for this activity in vitro, but the equivalent determinants have not been extensively assessed in vivo. Indeed, recent studies indicate differences between the in vitro and the in vivo degradation assays. We have performed an extensive analysis of the ability of a range of HPV-18 E6 mutants to direct p53 degradation in vivo. In addition, we have also compared the ability of HPV-18 E6 to direct the degradation of different oligomeric forms of p53 both in human and in murine cells. The results of these studies show that mutants of E6 exhibit very similar phenotypes both in vitro and in vivo. In contrast, mutants of p53 show markedly different susceptibilities in vitro and in vivo to E6-induced degradation, and this is further affected by the nature of the cell type in which the assays are performed. Finally, using a cell line temperature sensitive for the E1 ubiquitin-activating enzyme we have been able to show directly that this enzyme is involved in the process of E6-mediated degradation of p53 in vivo.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

Bacillus subtilis growing at 37 degrees C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37 degrees C is transferred to 20 degrees C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20 degrees C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20 degrees C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37 degrees C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.

A cassette containing a selectable cat gene and the lacZ gene without its own promoter has been incorporated into the mini-Mu bacteriophage genome. This mini-Mu derivative, referred to as mMu-Bs, can be used in Escherichia coli for the generation of lacZ transcriptional fusions to Bacillus subtilis genes cloned into plasmids. The resultant fusions can be analyzed in B. subtilis either as multicopy plasmids or as a single copy integrated via a Campbell-like recombination into the wild-type locus of the cloned fragment.

Storey et al. reply — These reports assess the frequency of the p53Arg allele in different populations and conclude that homozygous p53Arg is not a risk factor for cancer associated with human papilloma virus (HPV). The functional differences between the p53 isoforms that we have described1,2 provoked our initial epidemiological study. As we concluded then, it is crucial that investigations should be extended to different populations, and we are encouraged that such studies are underway.