Host-Targeted Antivirals Inhibit RACK1-mediated IRES Activities in HIV-1 Infection

Host ribosome-associated scaffold protein Receptor for Activated C Kinase 1 (RACK1) is utilized by a diverse group of human viruses for Internal Ribosomal Entry Sites (IRES) – mediated translation of viral mRNAs. We recently reported inhibition of herpes virus by small molecules targeting the RACK1 functional site. Here, we tested these molecules against HIV-1 and HCV, as HIV-1 contains two potential IRES sites and HCV translation occurs exclusively through IRES. Compounds significantly downregulated activities of HIV-1- and HCV-related dicistronic reporter constructs in transfected HEK293T cells. The compounds also strongly downregulated production of the HIV-1 capsid protein p24 in HIV-infected cells, as well as production of HIV-1 Gag precursor p55 and p55-derived proteins p24 and p17 in cells infected with the HIV-1 virus. Hepatitis C virus (HCV) IRES activities were also significantly inhibited by RACK1 inhibitor compounds. Since a number of human and plant pathogenic viruses are reported to use IRES, the RACK1 compounds can be established as broad host-targeted antivirals.

N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem–loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5′UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

Various miRNAs are involved in efficient HCV replication

One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5’UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5’UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure.Author summaryOne of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA, which is required for efficient propagation. Recently, it has been reported that interaction of miR-122 with the 5’UTR of HCV contributes to the folding of a functional IRES structure that is required for efficient translation of viral RNA. In this study, we examined the minimum motifs in the seed region of miRNAs required for the enhancement of HCV replication. As a result, we found two groups of non-hepatic miRNAs: “miR-122-like miRNAs” that can bind HCV-RNA at two locations in a manner similar to miR-122, and “non-miR-122-like miRNAs” that target a single site that masking both G28 and C29 in the 5’UTR. The interaction of these non-hepatic miRNAs with the 5’UTR can facilitate not only the folding of active HCV IRES but also the stabilization of IRES 3D structure in order to facilitate binding to the ribosome. These results suggest the possibility of replication of HCV in non-hepatic cells through interaction with miRNAs other than miR-122 and provide insight into the establishment of persistent infection of HCV in non-hepatic tissues that lead to the development of extrahepatic manifestations.

Syntheses and Binding Testing of N1-Alkylamino-Substituted 2-Aminobenzimidazole Analogues Targeting the Hepatitis C Virus Internal Ribosome Entry Site

A series of 2-aminobenzimidazole analogues have been synthesised and tested for binding to a previously established RNA target for viral translation inhibitors in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV). Synthesis of new inhibitor compounds followed a highly convergent strategy which allowed for incorporation of diverse tertiary amino substituents in high overall yields (eight-steps, 4–22%). Structure–activity relationship (SAR) studies focussed on the tertiary amine substituent involved in hydrogen bonding with the RNA backbone at the inhibitor binding site. The SAR study was further correlated with in silico docking experiments. Analogous compounds showed promising activities (half maximal effective concentration, EC50: 21–89µM). Structures of the synthesised analogues and a correlation to their mode of binding, provided the opportunity to explore parameters required for selective targeting of the HCV IRES at the subdomain IIa which acts as an RNA conformational switch in HCV translation.

Inhibition of EV71 replication by L3HYPDH, a newly identified interferon-stimulated gene product

ABSTRACTUp-regulation of interferon-stimulated genes (ISGs) is key to antiviral states mediated by interferon (IFN) but little is known about activity and underlying mechanisms of most ISGs against Enterovirus 71 (EV71). EV71 causes hand-foot-mouth disease in infants and occasionally severe neurological symptoms. Here we report that the product ofL3HYPDH, a newly identified ISG, inhibits the replication of EV71. This anti-EV71 activity was mapped to the C-terminal 60 amino acids region as well as the N-terminal region spanning from amino acid position 61 to 120 of L3HYPDH protein. L3HYPDH was shown to interfere with EV71 propagation at the RNA replication and protein translation levels. Specifically, L3HYPDH impairs translation mediated by the EV71 international ribosome entry site (IRES) but not by the HCV IRES, and this activity is conferred by the C-terminal region of L3HYPDH. Thus, L3HYPDH has antiviral activity against EV71, suggesting a potential mechanism for broad-spectrum antiviral effects of IFN.