A novel HBx genotype serves as a preoperative predictor and fails to activate the JAK1/STATs pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: Genetic variability in the hepatitis B virus X gene (HBx) is frequently observed and is associated with hepatocellular carcinoma (HCC) progression. However, a genotype classification based on the full-length HBx sequence and the impact of genotypes on hepatitis B virus (HBV)-related HCC prognosis remain unclear. We therefore aimed to perform this genotype classification and assess its clinical impact. METHODS: We classified the genotypes of the full-length HBx gene through sequencing and a cluster analysis of HBx DNA from a cohort of patients with HBV-related HCC, which served as the primary cohort (n = 284). Two independent HBV-related HCC cohorts, a validation cohort (n = 171) and a serum cohort (n = 168), were used to verify the results. Protein microarray assay analysis was performed to explore the underlying mechanism. RESULTS: In the primary cohort, the HBx DNA was classified into 3 genotypes: HBx-EHBH1, HBx-EHBH2, and HBx-EHBH3. HBx-EHBH2 (HBx-E2) indicated better recurrence-free survival and overall survival for patients with HCC. HBx-E2 was significantly correlated with the absence of liver cirrhosis, a small tumor size, a solitary tumor, complete encapsulation and Barcelona Clinic Liver Cancer (BCLC) stage A-0 tumors. Additionally, HBx-E2 served as a significant prognostic factor for patients with BCLC stage B HCC after hepatectomy. Mechanistically, HBx-E2 is unable to promote proliferation in HCC cells and normal hepatocytes. It also fails to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3)/STAT5 pathway. CONCLUSION: Our study identifies a novel HBx genotype that is unable to promote the proliferation of HCC cells and suggests a potential marker to preoperatively predict the prognosis of patients with BCLC stage B, HBV-associated, HCC. LAY SUMMARY: We classified a novel genotype of the full-length hepatitis B virus X gene (HBx), HBx-E2. This genotype was identified in tumor and nontumor tissues from patients with hepatitis B virus-related hepatocellular carcinoma. HBx-E2 could preoperatively predict the prognosis of patients with intermediate stage hepatocellular carcinoma, after resection.

Proteomic and phylogenetic coevolution analyses of pM79 and pM92 identify interactions with RNA polymerase II and delineate the murine cytomegalovirus late transcription complex.

The regulation of the late viral gene expression in betaherpesviruses is largely undefined. We have previously shown that the murine cytomegalovirus proteins pM79 and pM92 are required for late gene transcription. Here, we provide insight into the mechanism of pM79 and pM92 activity by determining their interaction partners during infection. Co-immunoprecipitation-coupled MS studies demonstrate that pM79 and pM92 interact with an array of cellular and viral proteins involved in transcription. Specifically, we identify RNA polymerase II as a cellular target for both pM79 and pM92. We use inter-protein coevolution analysis to show how pM79 and pM92 likely assemble into a late transcription complex composed of late transcription regulators pM49, pM87 and pM95. Combining proteomic methods with coevolution computational analysis provides novel insights into the relationship between pM79, pM92 and RNA polymerase II and allows the generation of a model of the multi-component viral complex that regulates late gene transcription.

Genetic characterization of natural variants of Vpu from HIV-1 infected individuals from Northern India and their impact on virus release and cell death.

BACKGROUND: Genetic studies reveal that vpu is one of the most variable regions in HIV-1 genome. Functional studies have been carried out mostly with Vpu derived from laboratory adapted subtype B pNL 4-3 virus. The rationale of this study was to characterize genetic variations that are present in the vpu gene from HIV-1 infected individuals from North-India (Punjab/Haryana) and determine their functional relevance. METHODS: Functionally intact vpu gene variants were PCR amplified from genomic DNA of HIV-1 infected individuals. These variants were then subjected to genetic analysis and unique representative variants were cloned under CMV promoter containing expression vector as well as into pNL 4-3 HIV-1 virus for intracellular expression studies. These variants were characterized with respect to their ability to promote virus release as well as cell death. RESULTS: Based on phylogenetic analysis and extensive polymorphisms with respect to consensus Vpu B and C, we were able to arbitrarily assign variants into two major groups (B and C). The group B variants always showed significantly higher virus release activity and exhibited moderate levels of cell death. On the other hand, group C variants displayed lower virus release activity but greater cell death potential. Interestingly, Vpu variants with a natural S61A mutation showed greater intracellular stability. These variants also exhibited significant reduction in their intracellular ubiquitination and caused greater virus release. Another group C variant that possessed a non-functional ß-TrcP binding motif due to two critical serine residues (S52 and S56) being substituted with isoleucine residues, showed reduced virus release activity but modest cytotoxic activity. CONCLUSIONS: The natural variations exhibited by our Vpu variants involve extensive polymorphism characterized by substitution and deletions that contribute toward positive selection. We identified two major groups and an extremely rare ß-TrcP binding motif mutant that show widely varying biological activities with potential implications for conferring subtype-specific pathogenesis.

Exploiting internal ribosome entry sites in gene therapy vector design.

Efficient and regulated co-expression of multiple genes is an important consideration in the design of gene therapy vectors. While the augmentation of a single therapeutic gene is often sufficient for gene therapy of simple mendelian disorders, strategies for the treatment of complex disorders and infectious diseases necessitate the introduction of multiple genes into the cell. Complex disorders such as cancer often involve mutations in multiple genes and a combination strategy targeting different defective genes simultaneously are often more effective than any single strategy. Likewise, approaches for treating infectious diseases such as HIV-1 (human immunodeficiency virus) often involve the blocking of multiple steps of the viral replication pathway simultaneously to prevent the emergence of resistant strains of the virus. Even for the treatment of single gene defects, the additional incorporation of a selectable marker gene is often necessary to achieve sustained expression of the therapeutic gene in the cells. Among the several different strategies to co-express multiple genes, the incorporation of an IRES (internal ribosome entry site) into gene therapy vector design represents one of the more promising strategies. IRES functions as a ribosome-landing pad for the efficient internal initiation of translation ensuring coordinate expression of several genes and are located at the 5'UTR (5' untranslated regions) of these genes. Currently, the most popular IRES utilized for gene therapy is the IRES from the EMCV (encephalomyocarditis virus). However, the major caveat with present vector systems utilizing this IRES is that the expression of the downstream gene is significantly less efficient than the upstream gene. This review will examine the growing list of naturally occurring and synthetic IRESes and how they can be exploited for human gene therapy.