Direct Interaction between Two Viral Proteins, the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex

Angel investor's selection criteria: a comparative institutional perspective

Despite the important role of angel investors as critical financial providers for new ventures, little is known regarding how institutions make their investment decisions. While angels make decisions based on selection criteria during the first stage, they are also embedded within and affected by different institutional settings and as a result weight these criteria differently than other investors. We compare angel investors' selection criteria in China and Denmark using the comparative institutional perspective. We use a policy capturing approach and hierarchy linear modeling, revealing that since Chinese angels are embedded within relationship-based institutional settings they tend to reply more on strong ties such as family and friends in management team, as well as weighting risks less compared to Danish angels operating within more rule-based institutional contexts

Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg.

Viral RNA synthesis was assayed in HeLa cells transfected with nonviable poliovirus RNA mutated in the genome-linked protein VPg-coding region. The transfecting RNA was transcribed in vitro from full-length poliovirus type 1 (Mahoney) cDNA containing a VPg mutagenesis cartridge. Hybridization experiments using ribonucleotide probes specific for the 3' end of positive- and negative-sense poliovirus RNA indicated that all mutant RNAs encoding a linking tyrosine in position 3 or 4 of VPg were replicated even though no virus was produced. VPg, but no VPg precursor, was found to be linked to the 5' end of the newly synthesized RNA. Encapsidated mutant RNAs were not found in transfected-cell lysates. After extended maintenance of transfected HeLa cells, a viable revertant of one of the nonviable RNAs was recovered; the revertant lost the lethal lesion in VPg by restoring the wild-type amino acid, but it retained all other nucleotide changes introduced during construction of the mutagenesis cartridge. Mutant RNA encoding phenylalanine or serine rather than tyrosine, the linking amino acid in VPg, was not replicated in transfected cells. A chimeric mutant containing the VPg-coding region of coxsackievirus within the poliovirus genome was viable but displayed impaired multiplication. A poliovirus-coxsackievirus chimera lacking a linking tyrosine in VPg was nonviable and replication-negative. The results indicate that a linkage-competent VPg is necessary for poliovirus RNA synthesis to occur but that a step in poliovirus replication other than initiation of RNA synthesis can be interrupted by lethal mutations in VPg

CARDIOVIRAL INTERNAL RIBOSOMAL ENTRY SITE IS FUNCTIONAL IN A GENETICALLY ENGINEERED DICISTRONIC POLIOVIRUS

HIGH mutation rates have driven RNA viruses to shorten their genomes to the minimum possible size 1. Mammalian (+)-strand RNA viruses and retroviruses have responded by reducing the number of cis-acting regulatory elements, a constraint that has led to the emergence of the polyprotein 2. Poliovirus is a (+)-stranded picornavirus whose polyprotein, encoded by an open reading frame spanning most of the viral RNA 3, is processed by virus-encoded proteinases 4,5. Despite their genetic austerity, picornaviruses have retained long 5' untranslated regions 6-8, which harbour cis-acting elements that promote initiation of translation independently of the uncapped 5' end of the viral messenger RNA 9-12. These elements are termed 'internal ribosomal entry sites' 10 and are formed from highly structured RNA segments 13-15 of at least 400 nucleotides 16. How these elements function-is not known, but special RNA-binding proteins may be involved 17. The ribosome or its 40S subunit probably binds at or near a Y(n)X(m)AUG motif (where Y is a pyrimidine and X is a purine) at the 3' border of the internal ribosomal entry site 17, which either provides the initiating codon 16,18 or enables the ribosome to translocate to one downstream (E.W. et al., submitted). Initiation from most eukaryotic messenger RNAs usually occurs by ribosomal recognition of the 5' and subsequent scanning to the AUG codon 19. Here we describe a genetic strategy for the dissection of polyproteins which proves that an internal ribosomal entry site element can initiate translation independently of the 5' end.X11101sciescopu