We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism is not clear. Here, it is reported that influenza A virus NS1 protein is responsible for PI3K/Akt pathway activation. It was demonstrated that the NS1 protein interacts with the p85 regulatory subunit of PI3K via direct binding to the SH3 and C-terminal SH2 domains of p85. Consensus binding motifs for SH3 and SH2 domains were found in influenza A virus NS1, namely an SH2-binding motif (YXXXM) at aa 89, SH3-binding motif 1 (PXXP) around aa 164 and SH3-binding motif 2 around aa 212. Mutant virus encoding NS1 protein with mutations in the SH-binding motifs failed to interact with SH domains of p85 and did not activate the PI3K/Akt pathway. The mutant virus is attenuated in Madin-Darby canine kidney cells. Our study has established a novel function of NS1: by interacting with p85 via the SH-binding motifs, NS1 can activate the PI3K/Akt pathway.RNA segment 8 of Influenza A virus encodes two proteins, NS1 and NS2/NEP (Lamb, 1989). NS1 is a multifunctional protein that is translated from unspliced mRNA (Lamb & Choppin, 1979). Two functional domains have been identified in the NS1 protein: an RNA-binding domain near the N terminus and an effector domain in the C terminus . The RNA-binding activity of the NS1 protein correlates to its ability to inhibit cellular pre-mRNA splicing (Lu et al., 1994;Qiu et al., 1995). It is also linked to the ability to counteract cellular alpha/beta interferon functions efficiently (Garcia-Sastre et al., 1998) by inhibiting the activation of the protein kinase PKR (Lu et al., 1995;Hatada et al., 1999) and transcription factors NF-kB, IRF-3 and IRF-7 (Talon et al., 2000;Wang et al., 2000). Within the effector domain, two binding sites of cellular proteins were identified. The binding site for CPSF is positioned around aa 186 and the PABII-binding site is located between aa 223 and 237 (Li et al., 2001). These binding sites are required for the inhibition of 39-end processing of cellular pre-mRNAs; thus, they are important for influenza virus replication (Noah et al., 2003).Phosphatidylinositol 3-kinases (PI3Ks) are a family of cellular, heterodimeric enzymes that consist of a regulatory subunit (p85) and a catalytic subunit (p110). PI3K is activated by binding of the SH domain in the p85 subunit to autophosphorylated tyrosine kinase receptors, or to non-receptor tyrosine kinases or some viral proteins in the cytoplasm (Carpenter et al., 1993;Street et al., 2004). After activation, the p110 subunit of PI3K phosphorylates the lipid substrate phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (Toker & Cantley, 1997). This molecule serves as a lipid second messenger and is able to regulate phosphorylation of a number of kinases, including Akt. Akt is activated via phosphorylation at Thr308 and Ser473 (Alessi et al., 1996). Phosphorylated Akt plays a central role in modulating diverse downstream signalling pathways associated with cell survival, pro...

The phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has attracted much recent interest due to its central role in modulating diverse downstream signalling pathways associated with cell survival, proliferation, differentiation, morphology and apoptosis. An increasing amount of information has demonstrated that many viruses activate the PI3K/Akt pathway to augment their efficient replication. In this study, the effect of the PI3K/Akt signalling pathway on influenza virus propagation was investigated. It was found that Akt phosphorylation was elevated in the late phase of influenza A/PR/8/34 infection in human lung carcinoma cells (A549). The PI3K-specific inhibitor LY294002 could suppress Akt phosphorylation, suggesting that influenza A virus-induced Akt phosphorylation is PI3K-dependent. UV-irradiated influenza virus failed to induce Akt phosphorylation, indicating that viral attachment and entry were not sufficient to trigger PI3K/Akt pathway activation. Blockage of PI3K/Akt activation by LY294002 and overexpression of the general receptor for phosphoinositides-1 PH domain (Grp1-PH) led to a reduction in virus yield. Moreover, in the presence of LY294002, viral RNA synthesis and viral protein expression were suppressed and, possibly as a consequence of low NP and M1 protein level, viral RNP nuclear export was also suppressed. These data suggest that the PI3K/Akt signalling pathway plays a role in influenza virus propagation.

Recent studies have demonstrated that influenza

Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using in vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation. IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.