Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

人CTLA4Ig和EGFP双基因共表达逆转录病毒载体构建及鉴定

目的 构建并鉴定人CTLA4Ig和增强型绿色荧光蛋白（EGFP）双基因共表达逆转录病毒载体。方法 利用基因重组技术将IRES序列与CTLA4Ig和EGFP基因构建到逆转录病毒载体中，脂质体法将重组质粒pCTLA4Ig-IRES2-EGFP转染PA317细胞，在G418选择压力下，通过流式细胞检测筛选得到高效共表达CTLA4Ig和EGFP并能产生高滴度重组逆转录病毒的PA317细胞克隆，MTT法测定CTLA4Ig生物学活性。结果 成功构建出含IRES序列的CTLA4Ig和EGFP双基因共表达逆转录病毒载体，生物学活性测定表明CTLA4Ig生物学活性没有受到影响。结论 IRES序列调控CTLA4Ig与EGFP双基因在细胞中同时独立表达。     

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin,FGF2, and SERCA2a into Ischemic Heart

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Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Functional conservation despite structural divergence in ligand-responsive RNA switches

An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.Internal ribosome entry site (IRES) elements provide an alternative mechanism for translation initiation by directing the assembly of functional ribosomes directly at the start codon in a process that does not require 5′ cap recognition or ribosomal scanning and that is independent of many host initiation factors (1–4). The genomes of Flaviviridae and Picornaviridae contain elements that share similarity with the archetypical hepatitis C virus (HCV) IRES in overall domain organization, but not sequence or details of secondary structure (5). The HCV IRES adopts a complex architecture of four independently folding domains (Fig. 1A) (6). Domain II is nearly 100% conserved in clinical isolates (7) and has analogous counterparts in other viral IRES elements, all of which display some secondary structure similarity, but significant sequence variation in the subdomain IIa-like internal loop (Fig. 1B). Domain II has been shown to promote stable entry of HCV and classic swine fever virus (CSFV) mRNA at the decoding groove of the 40S subunit (8–10) and is required for initiation factor removal before ribosomal subunit joining (11), as well as adjustment of initiator tRNA orientation (12). The transition from initiation to elongation stages of translation depends critically on domain II (13). Recently, direct interaction of HCV domain II with initiator tRNA has been demonstrated (14). In HCV, subdomain IIa folds into an L-shaped motif (15) (Fig. 1C) that introduces a 90° bend in domain II (16) and directs the IIb hairpin toward the E-site at the ribosomal subunit interface (17, 18).Open in a separate windowFig. 1.Structures and ligands of viral IRES. (A) The IRES in the 5′ UTR of the HCV genome. The location of subdomain IIa is highlighted by an orange box. The viral genome encodes structural (S) and nonstructural (NS) proteins and contains a structured 3′ UTR. (B) Secondary structure predictions of domain II motifs in viral IRES elements from HCV and other flaviviruses, including CSFV and BVDV, as well as picornaviruses such as AEV and SVV. Non-Watson–Crick base pairs are indicated by the ○ symbol. Sequence conservation is indicated in red. (C) Crystal structure of the subdomain IIa RNA from HCV. (D) Benzimidazole (1) and diaminopiperidine (2) inhibitors of IRES-driven translation that target the HCV subdomain IIa. (E) Crystal structure of the HCV subdomain IIa RNA in complex with inhibitor 1.The HCV IRES subdomain IIa is the target for viral translation inhibitors (Fig. 1D) that bind to the internal loop and block translation by capturing distinct conformational states of the RNA (7). Structure analysis revealed that benzimidazole inhibitors such as compound 1 (19, 20) interact with an extended architecture of IIa in which the stems flanking the internal loop are coaxially stacked on both sides of the ligand-binding pocket (Fig. 1E) (21). In contrast, diaminopiperidine compounds such as 2 bind and lock the IIa RNA in a bent conformation that corresponds to the ligand-free state (22). Conformational capture of the subdomain IIa switch by ligands in solution was demonstrated by FRET experiments and established as a mechanism of IRES inhibition (23). On the basis of these findings, it was proposed that subdomain IIa may be the target for a cognate biological ligand whose adaptive recognition by the RNA motif may facilitate ribosome release from the IRES-bound complex (7).Here, we have explored potential candidates for a cognate ligand of the subdomain IIa switch and investigated the structural and functional conservation of similar ligand responsive switch motifs in other IRES RNAs.     

Hepatitis C infection of B lymphocytes: more tools to address pending questions

Evaluation of: Durand T, Di Liberto G, Colman H et al. Occult infection of peripheral B cells by hepatitis C variants which have low translational efficiency in cultured hepatocytes. Gut 59, 934–942 (2010).

Hepatitis C virus (HCV) infection spreads primarily via contact with infected blood and can establish a persistent infection in 80% of infected individuals, progressively causing chronic liver disease that can lead to hepatocellular carcinoma or end-stage liver disease requiring a transplant. There is no vaccine, and current treatment with interferon and ribavirin is costly, poorly tolerated and ineffective for a large proportion of patients. Technical limitations have stifled the study of HCV immunology, and hence the correlates of resolution remain elusive. HCV robustly infects hepatocytes in the liver, yet HCV RNA is often found to be associated with peripheral blood lymphocytes and extrahepatic manifestations of the disease include B-cell abnormalities. The few existing characterized viral clones that can replicate in vitro have consistently failed to infect immune cells; however, some groups have detected low levels of replication in peripheral blood cells, hinting that occult forms of infection may be possible. HCV lymphotropism remains a controversial subject that needs to be elucidated in order to identify viral reservoirs that may provide targets for therapeutic intervention. The precise interactions between HCV and immune cells need to be determined to establish if the virus has developed mechanisms to modulate immune responses. In the study by Durand et al., correlations were sought between cell tropism and mutations in the 5´ noncoding region of the HCV genome, known as the internal ribosome entry site. Key findings are discussed here, highlighting current experimental challenges that surround the topic of HCV lymphotropism.     

Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-kappaB and STAT-3

Human hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. The HCV RNA genome codes for at least ten proteins. The HCV non-structural protein 5A (NS5A) has generated considerable interest due to its effect on interferon sensitivity via binding and inactivating the cellular protein kinase, PKR. It has been shown that NS5A engages in the endoplasmic reticulum (ER)-nucleus signal transduction pathway. The expression of NS5A in the ER induces an ER stress ultimately leading to the activation of STAT-3 and NF-kappaB. This pathway is sensitive to inhibitors of Ca(2+) uptake in the mitochondria (ruthenium red), Ca(2+) chelators (TMB-8, EGTA-AM), and antioxidants (PDTC, NAC, Mn-SOD). The inhibitory effect of protein tyrosine kinase (PTK) inhibitors indicates the involvement of PTK in NF-kappaB activation by NS5A. This implicates an alternate pathway of NF-kappaB activation by NS5A. The actions of NS5A have also been studied in the context of an HCV subgenomic replicon inducing a similar intracellular event. Thus, activation of NF-kappaB leads to the induction of cellular genes, which are largely antiapoptotic in function. These studies suggest a potential function of NS5A in inducing chronic liver disease and hepatocellular carcinoma associated with HCV infection.