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.

A new Dicistrovirus from soldier fly Inopus flavus (James) (Diptera: Stratiomyidae), a pest of sugarcane

The native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. While adult soldier flies do not feed on sugarcane, larvae spend one to two-years underground feeding on roots, causing mechanical and systemic damage to crops (Saccharum officinarum L.) that impacts yield. Current measures of pest control commonly target above ground pests and are ineffective against solider fly larvae, highlighting the importance of novel control methods. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the Dicistroviridae family. Viruses from this family have been found naturally infecting insects from a range of taxonomic groups and they often cause pathogenesis in their hosts. To characterise the genetic and physical properties of the new virus, the positive RNA genome was analysed using a combination of sequencing approaches. The virus genome is organised similarly to members of the Dicistroviridae with two open reading frames (ORF) the first encoding non-structural proteins and the second encoding structural proteins. The genome includes two potential internal ribosomal entry sites (IRES) one within the 5’ UTR and the other in the intergenic region (IGR). Based on the amino acid sequences of the non-structural and structural polyproteins encoded by the two ORF soldier fly virus groups within the dicistrovirus family. Virus particles purified from infected larvae and visualised by electron microscopy are icosahedral, non-enveloped, and 30 nm in diameter. The genetic and physical characteristics of this novel soldier fly virus are consistent with it being a member of the Dicistroviridae.

The Halastavi árva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

ABSTRACTDicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate

Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.

IRES-mediated cap-independent translation, a path leading to hidden proteome

Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry sites (IRESs) can serve as an alternative mechanism for protein production. Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trans-acting factors. Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood, the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities. A recent screen identified > 1000 putative IRESs from viral and human mRNAs, expanding the scope and mechanism for cap-independent translation. Additionally, a large number of circular RNAs lacking free ends were identified in eukaryotic cells, many of which are found to be translated through IRESs. These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA, implying a hidden proteome produced from cap-independent translation.