Activated Gab1 drives hepatocyte proliferation and anti-apoptosis in liver fibrosis via potential involvement of the HGF/c-Met signaling axis.

Chronic liver diseases are caused by hepatic viral infection, chemicals, and metabolic stress. The protein Grb2-associated binder 1 (Gab1) binds to various growth factor receptors, and triggers cell differentiation/survival signaling pathways. To identify signaling molecules involved in the progression of liver diseases, we performed reverse-phase protein microarray (RPMA)-based screening of hepatocytes isolated from humanized mice after acute HCV infection. Acute viral infection in humanized liver mice significantly decreased the level of hepatocyte p-Gab1. Moreover, hepatoma cells upon HCV infection decreased Gab1 mRNA at later times of infection (D3 to D5) and p-Gab1 level was inversely related to the production of TGF-ß. In contrast, the level of p-Gab1 was increased in CCL4-induced fibrotic liver. Hepatoma cells showed elevation of p-Gab1, along with an increase in STAT3 and ERK activation, upon treatment with HGF (ligand of HGF receptor/c-Met) and CCL4. In Gab1 knockdown hepatoma cells, cell proliferative signaling activity was reduced but the level of activated caspase-3 was increased. These findings suggest that hepatocyte Gab1 expression may play a role in promoting liver fibrosis progression by triggering ERK activation and inhibiting apoptosis. It implies that the Gab1-mediated signaling pathway would be a promising therapeutic target to treat chronic liver diseases.

Hepatitis C Virus Dysregulates Polyamine and Proline Metabolism and Perturbs the Urea Cycle.

Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.

Bystander Effects and Profibrotic Interactions in Hepatic Stellate Cells during HIV and HCV Coinfection.

This study aims to explore the influence of coinfection with HCV and HIV on hepatic fibrosis. A coculture system was set up to actively replicate both viruses, incorporating CD4 T lymphocytes (Jurkat), hepatic stellate cells (LX-2), and hepatocytes (Huh7.5). LX-2 cells' susceptibility to HIV infection was assessed through measurements of HIV receptor expression, exposure to cell-free virus, and cell-to-cell contact with HIV-infected Jurkat cells. The study evaluated profibrotic parameters, including programed cell death, ROS imbalance, cytokines (IL-6, TGF-ß, and TNF-α), and extracellular matrix components (collagen, α-SMA, and MMP-9). The impact of HCV infection on LX-2/HIV-Jurkat was examined using soluble factors released from HCV-infected hepatocytes. Despite LX-2 cells being nonsusceptible to direct HIV infection, bystander effects were observed, leading to increased oxidative stress and dysregulated profibrotic cytokine release. Coculture with HIV-infected Jurkat cells intensified hepatic fibrosis, redox imbalance, expression of profibrotic cytokines, and extracellular matrix production. Conversely, HCV-infected Huh7.5 cells exhibited elevated profibrotic gene transcriptions but without measurable effects on the LX-2/HIV-Jurkat coculture. This study highlights how HIV-infected lymphocytes worsen hepatic fibrosis during HCV/HIV coinfection. They increase oxidative stress, profibrotic cytokine levels, and extracellular matrix production in hepatic stellate cells through direct contact and soluble factors. These insights offer valuable potential therapies for coinfected individuals.

PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.

Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

RNA-binding proteins and exoribonucleases modulating miRNA in cancer: the enemy within.

Recent progress in the investigation of microRNA (miRNA) biogenesis and the miRNA processing machinery has revealed previously unknown roles of posttranscriptional regulation in gene expression. The molecular mechanistic interplay between miRNAs and their regulatory factors, RNA-binding proteins (RBPs) and exoribonucleases, has been revealed to play a critical role in tumorigenesis. Moreover, recent studies have shown that the proliferation of hepatocellular carcinoma (HCC)-causing hepatitis C virus (HCV) is also characterized by close crosstalk of a multitude of host RBPs and exoribonucleases with miR-122 and its RNA genome, suggesting the importance of the mechanistic interplay among these factors during the proliferation of HCV. This review primarily aims to comprehensively describe the well-established roles and discuss the recently discovered understanding of miRNA regulators, RBPs and exoribonucleases, in relation to various cancers and the proliferation of a representative cancer-causing RNA virus, HCV. These have also opened the door to the emerging potential for treating cancers as well as HCV infection by targeting miRNAs or their respective cellular modulators.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

Association of genotypes with viral load and biochemical markers in HCV-infected Sindhi patients

Abstract The presented study had two objectives. The first was to examine distributions of Hepatitis C Virus (HCV) genotypes in Sindh, Pakistan, where HCV is prevalent. The other was to explore clinically relevant relationships between the genotypes, viral load (measured by real-time polymerase chain reaction assays) and biochemical markers. For this, 1471 HCV-infected patients in six cities in Sindh were recruited and sampled. HCV genotype distributions varied among the cities, but genotype 3a was most prevalent, followed by 3b, 1a and 1b (detected in 51.5, 22.7. 9.25 and 3.2% of the cases, respectively). No type-specific sequences were detected in serum samples from 189 (12.8%) of the 1471 patients. Frequencies of low (<200,000 IU/mL serum), intermediate (200,000-600,000 IU/mL serum) and high (>600,000 IU/mL serum) viral loads were respectively 45.4, 16.5 and 38.1% for patients infected with genotype 3, and 16.9, 36.9 and 46.2%, respectively, for patients with other genotypes. Infection with genotype 1a was associated with significantly higher (p < 0.005) alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase titers than infection with genotype 3a. The results will help in the formulation of treatment strategies.

Análise de quasiespécies do vírus da hepatite C (HCV) e implicação na transmissão intrauterina / Analysis of quasispecies of the hepatitis C virus (HCV) and involvement in transmitting utero

A transmissão materno-infantil (TMI) é a causa mais comum de infecção pelo vírus da hepatite C (HCV) entre as crianças. Objetivo: Esse estudo teve como objetivo avaliar fatores virais implicados na TMI do HCV. Materiais e métodos: Quatro gestantes e um par mãe-recém-nascido (RN), todos infectados pelo HCV, foram incluídos neste estudo. Sequências das regiões 5’UTR, E1, HVR1, E2 e NS5B foram obtidas através de sequenciamento direto do produto do PCR e clonagem. A diversidade quasiespécie foi analisada utilizando-se diferentes parâmetros (taxa de clonotipos, frequência de mutações, Pn e entropia de Shannon normalizada), comparando (1) grupos TMI+ e TMI-, e (2) par mãe-RN. Um framework foi usado para avaliar a associação entre a frequência dos nucleotídeos e a TMI. Resultados: Dois casos de TMI foram identificados, mas apenas a amostra de um RN estava disponível. As cargas virais de todos os sujeitos estavam acima do limite de quantificação. Ambos os casos de TMI pertenciam ao genótipo 1a apenas este subtipo foi analisado subsequentemente. O sequenciamento direto dos produtos de PCR não representou, de maneira confiável, a complexidade quasiespécie e não foi utilizado. Não houve clonotipos coincidentes entre os grupos TMI+ e TMI-, exceto pela região 5’UTR. Em nível de aminoácido, mãe e RN compartilharam apenas do clonotipo predominante. Todos os clonotipos minoritários foram exclusivos. Foi observada maior diversidade quasiespécie nas regiões E2 e NS5B. A HVR1 apresentou a menor diversidade dentro da região codificante. A diversidade quasiespécie do grupo TMI+ foi sempre maior do que aquela vista no grupo TMI-; no entanto, não houve significância estatística. Trinta e cinco mutações na região codificante foram associadas significativamente com a TMI. Dados do par mãe-RN sugerem que a transmissão intrauterina ocorreu em um momento inicial da gestação e que o vírus provavelmente atravessou o tecido placentário, levando a um gargalo de garrafa. Conclusões: A diversidade quasiespécie não foi associada à TMI, mas a presença de mutações ao longo da região codificante sugere que o genoma completo contribui para a capacidade de transmissão intrauterina. São necessários estudos adicionais para determinar se essas variantes podem ser úteis para predizer a TMI.

Carcinoma hepatocelular na adolescência: relato de caso / Hepatocellular carcinoma in adolescence: case report

O carcinoma hepatocelular é uma das malignidades mais comuns do fígado. Atualmente sua incidência estáaumentando a nível mundial com perspectivas de um maior número de casos para os próximos anos. A maiorparte dos acometidos é do sexo masculino e com faixa etária acima dos cinqüenta anos de idade. Sabe-se queo risco para o desenvolvimento do carcinoma hepatocelular está intimamente associado às hepatites virais crônicas, alcoolismo crônico, cirrose, doenças metabólicas e genéticas e outros fatores de risco. A ausência de sintomas na fase inicial desta patologia torna o diagnósticomais comum em fases avançadas, através de exames de imagem. Há diversas opções terapêuticas à disposição, sendo a hepatectomia parcial, radical e o transplante hepático as medidas com potencial curativo. Relata-se o caso de uma paciente com 15 anos que iniciou o quadro com dor e aumento do volume abdominal. A Ainvestigação realizada não encontrou fatores de risco para a doença e o diagnóstico foi realizado através deexames de imagem. A doença já se encontrava metastática no momento do diagnóstico. A paciente foi tratadapaliativamente, evoluindo para óbito dois meses após o diagnóstico.

Hepatocellular carcinoma is one of the most common malignancies of the liver. Currently, its incidence is rising around the globe and perspectives of larger amounts of cases are expected for the next years. Most of the patients are males about fifty-years-old. It's known that the risk of developing hepatocellular carcinoma is associatedto chronic viral hepatitis, chronic alcoholism, cirrhosis, metabolic and genetic diseases and many othersrisk factors. The absence of symptoms in the early stages of this disease may lead to diagnosis in late stages, through imaging tests. There are many therapeutical options available. Partial and radical hepatectomy and hepatic transplant have curative potential. It's reported the case of a 15-years-old patient with abdominal pain and increase of abdominal volume. No risk factors for the disease were found and the diagnosis was made thoughimaging tests. The disease was already metastatic at the time of the diagnosis. The patient was treated with paliative care and died two months after the diagnosis.

Detecção imunoistoquímica das oncoproteínas p21ras, c-myc E p53 no carcinoma hepatocelular e no tecido hepático não-neoplásico / Immunohistochemical detection of p21ras, c-myc and p53 oncoproteins in hepatocellular carcinoma and in non-neoplastic liver tissue

RACIONAL: A hepatocarcinogênese é um processo no qual as alterações genéticas e epigenéticas são bem conhecidas em modelos animais, mas carece de estudos no homem. OBJETIVOS: Analisar a freqüência das oncoproteínas p21ras, c-myc e p53 no carcinoma hepatocelular e no fígado não-neoplásico. Verificar ainda a associação destas oncoproteínas com os padrões e graus histológicos, assim como com as infecções pelos vírus das hepatites B e C. MÉTODOS: Foi analisada por método imunoistoquímico a detecção das oncoproteínas p21ras, c-myc e p53 em 47 casos de carcinoma hepatocelular e no tecido não-neoplásico circunjacente ao tumor (40 casos). RESULTADOS: As oncoproteínas p21ras, c-myc e p53 foram detectadas, respectivamente, em 44,7 por cento, 53,2 por cento e 36,2 por cento dos casos de carcinoma hepatocelular. A imunorreatividade do p21ras e c-myc mostrou uma associação significativa. Contudo, não houve associação significativa entre a detecção do p21ras, c-myc e p53 com os diferentes graus e padrões histológicos, nem tampouco com as infecções pelos vírus das hepatites B e C. A mesma associação significativa entre o p21ras e c-myc foi encontrada no tecido não-neoplásico dos casos de cirrose em relação aos que não apresentaram cirrose, enquanto que o p53 foi negativo em todos os casos. CONCLUSÕES: A imunorreatividade das oncoproteínas p21ras, c-myc e p53 corrobora evidências prévias de sua detecção no carcinoma hepatocelular, o que sugere poder haver participação destas proteínas na hepatocarcinogênese humana. A significativa associação entre as proteínas p21ras, c-myc e p53 no carcinoma hepatocelular e na cirrose pode apontar uma interação entre as mesmas, sobretudo na hepatocarcinogênese pela via da cirrose.

Perfiles de glucosa e insulina en pacientes con hepatitis crónica por virus C / Glucose and insulin profiles on patients with chronic C hepatitis

Con objeto de estudiar el comportamiento de los perfiles de glucosa e insulina en pacientes con hepatitis crónica por virus C (HCV), se incluyeron once pacientes con edad promedio de 47.5 años e índice de masa corporal de 23.8 por ciento 1.4. Cinco de ellos eran diabéticos. Su diabetes apareció años después de la hepatitis. El grupo control estuvo compuesto por 12 sujetos sanos con edad promedio de 42.8 años e índice de masa corporal de 24.1 ñ 1.2. Los pacientes tuvieron hepatitis crónica con diferentes grados de daño hepático, pero sin cirrosis. Las cifras de glucosa en ayunas fueron 119.9 ñ 43.4 mg/dL para el grupo de pacientes y 91.9 ñ 3.6 mg/dL para el control. Los niveles de insulina de ayunas fueron 28.1 ñ 17 µU/mL para el grupo de pacientes y 12.9 ñ 3.9 µU/mL para el control. Los valores posprandiales de insulina fueron 38.5 ñ 38 µU/mL para el grupo de pacientes y 51.04 ñ 27 µU/mL para el control. Los valores poprandiales de glucosa fueron 146.9 ñ 118 mg/dL para el grupo de pacientes y 104 ñ 15 mg/mL para el control. En cuanto a la glucosa y a la insulina, el grupo de pacientes no mostró diferencias significativas cuando sus valores de ayuno se compararon versus los posprandiales, contrario a lo que sucedió en el control (p < 0.01 y p< 0.005). Se sugiere que en pacientes con HCV se efectúen periódicamente mediciones de glucemia de ayuno y posprandiales para detectar oportunamente alteraciones de hiperglucemia