Functional Characterization of the Interaction between Human La and Hepatitis B Virus RNA

Abstract

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.RNA metabolism depends on the formation of ribonucleoprotein particles mediating diverse processes such as splicing, polyadenylation, nuclear export, and the regulation of mRNA stability (1, 2). The formation of RNPs 1 is a tightly controlled process, potentially regulated by several stimuli, including hormones and cytokines. Such stimuli can alter the RNA binding activity of proteins on the post-translational level by phosphorylation or dephosphorylation and thereby the processing and stability of RNAs (3-5). In addition, RNA processing depends on various cis-acting elements including splice sites, export elements, and endoribonucleolytic cleavage sites recognized by RNA-binding proteins. To understand fully the regulation of processing of a specific RNA, both trans-acting factors and cis-acting elements as well as their functions need to be known. The same applies for a detailed understanding of the metabolism of viral RNA. Such studies could lead to the identification of novel cellular targets valuable for the development of innovative antiviral strategies when focused on the post-transcriptional control of RNAs of viruses with global medical importance. This applies to hepatitis B virus (HBV) with more than 300 million chronically infected carriers worldwide who await more effective antiviral therapies.HBV is a noncytopathic, hepatotropic virus with a 3.2-kb circular DNA genome. After conversion into a covalently closed circular DNA, this genome serves in the nucleus as a template for transcription of all viral RNAs. Synthesis of these transcripts is driven by at least four promotors, leading to a large size heterogeneity with many different 5Ј-ends, whereas they all have very similar 3Ј-ends because of processing at the same polyadenylylation site (6). The so-called pregenomic RNA (slightly longer than genome length) is encapsidated into nucleocapsids where it is reverse-transcribed into viral DNA. This RNA serves also as messenger for synthesis of the viral P protein as well as for the core protein. The viral surface proteins as well as a regulatory protein with a role in hepatocarcinogenesis, des...

Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs

“…It was reported previously that the La protein contributes to HBV pregenomic RNA stability through specific binding to the viral RNA, while cytotoxic T-lymphocyte (CTL) and interleukin-2 (IL-2) treatment results in the fragmentation of the La protein, which renders viral RNA vulnerable to degradation by cellular nucleases (5,13,14,16,44). To determine whether MyD88 induces the fragmentation of the La protein, we studied the expression of the La protein in MyD88-overexpressing cells by Western blot analysis.…”

“…For these plasmids, the synthesis of the pregenomic RNA is driven by the cytomegalovirus (CMV) promoter and the Tet promoter, respectively. pCMV-HBV M2 is a derivative of pCMV-HBV in which the La protein-binding sites have been mutated (5). pCMV-HBV⌬PRE is a PRE deletion mutant of pCMV-HBV (15).…”

The RNA-binding protein La contributes to cell proliferation and CCND1 expression

“…In addition to the well-described functions of the RNA-binding protein La in the processing of RNA polymerase III transcripts (for reviews, see Maraia, 2001;Wolin and Cedervall, 2002), the mammalian La interacts with both cellular (McLaren et al, 1997;Brenet et al, 2005) and viral mRNAs (Spangberg et al, 2001;Ehlers et al, 2004), and many reports suggest a role of La in regulation of cellular and viral translation. For instance, human La supports internal ribosome entry site (IRES)-and cap-dependent translation of viral mRNAs, such as hepatitis C virus, polio virus (Ali et al, 2000;Costa-Mattioli et al, 2004;Pudi et al, 2004), HIV TAR mRNA (Svitkin et al, 1994;Chang et al, 1995), suppresses translation of the hepatitis A virus (Cordes et al, 2008), and regulates IRES-and cap-dependent translation of cellular mRNAs (for example, BiP mRNA , X-linked inhibitor of apoptosis protein (Holcik and Korneluk, 2000), 5 0 TOP-mRNAs (Crosio et al, 2000) and Mdm2 mRNA (Trotta et al, 2003)).…”

Indoleamine 2,3-Dioxygenase Mediates the Antiviral Effect of Gamma Interferon against Hepatitis B Virus in Human Hepatocyte-Derived Cells

“…However, we did not observe any cytotoxic effect in HepG2 cultures transfected with a plasmid expressing IDO (data not shown). In addition, the fact that overexpression of IDO does not affect the steady-state level of HBV pgRNA, for which the half-life is less than 12 h in cell cultures (11,68,69), plus the lack of effect on the steady-state content of cellular proteins, such as TDO and ␤-actin (Fig. 4, bottom, lane 3), strongly suggested that expression of IDO in HepG2 cells did not significantly affect the host cellular transcription and translation machinery but preferentially inhibited HBV protein and DNA biosynthesis.…”