A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5Ј untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5Ј UTR/ luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant downregulation of HCV 5Ј UTR/luciferase reporter expression (range 40% to 80% inhibition, P F .05). Moreover, several ribozymes showed significant inhibition (G90%, Chronic infection with hepatitis C virus (HCV) can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. 1,2 The Centers for Disease Control recently reported the number of chronically infected Americans to be approximately 4.5 million; thus, HCV infection is over four times as prevalent as human immunodeficiency virus infection. 3 Worldwide the prevalence of chronic HCV is similar to that found in the United States. 3 Thus, chronic HCV infection represents an important public health problem throughout the world.PTreatment of chronic HCV infection with interferon alfa leads to sustained viral clearance in only approximately 12% of patients. 4 Newer therapeutic regimes, such as the combination of interferon alfa and ribavirin, can lead to 38% to 43% of patients having a sustained virological response. 5,6 However, even with current combination regimes, approximately 60% of patients have no sustained virological benefit. Additionally, treatment with interferon alfa and ribavirin leads to significant toxicities. 5,6 Therefore, there still remains a great need for improved therapeutic modalities.HCV is a 9.5-kb, plus-strand, RNA virus that is a member of the human flavivirus family. 7,8 Although the sequence of the HCV-RNA genome is highly variable among clinical isolates, the 5Ј untranslated region (UTR) of the genome is highly conserved with respect to RNA sequence identity. 9,10 The conserved sequence/structure of the 5Ј UTR of HCV RNA contains an internal ribosome entry site (IRES) to mediate translation independent of a 5Ј-cap structure. 10,11 The HCV IRES does not require any viral protein for initiation of translation 12 and IRES elements also occur in cellular messenger RNAs. 13,14 Because the components of IRES-mediated translation are shared between cellular and HCV messenger RNAs, targeting the mechanism of HCV-IRES-mediated translation can be problematic. However, because the HCV-IRES sequence is highly conserved among viral genotypes, it is an excellent target for ribo...

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus Bi in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, a-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP7O-Pl complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.