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Biomedical subjects

C U Hellen

Publications and source records attributed to C U Hellen.

13 recordsLinked to original sources
PubMed1992

The role of proteolytic processing in the morphogenesis of virus particles.

Proteinases are encoded by many RNA viruses, all retroviruses and several DNA viruses. They play essential roles at various stages in viral replication, including the coordinated assembly and maturation of virions. Most of these enzymes belong to one of three (Ser, Cys or Asp) of the four major classes of proteinases, and have highly substrate-selective and cleavage specific activities. They can be thought of as playing one of two general roles in viral morphogenesis. Structural proteins are encoded by retroviruses and many RNA viruses as part of large polyproteins. Their proteolytic release is a prerequisite to particle assembly; consequent structural rearrangement of the capsid domains serves to regulate and direct association and assembly of capsid subunits. The second general role of proteolysis is in assembly-dependent maturation of virus particles, which is accompanied by the acquisition of infectivity.

Endopeptidases↗
PubMed1992

Viral proteases as targets for chemotherapeutic intervention.

Many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. The biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. Techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases.

Amino Acid Sequence↗
PubMed1992

A 1.5 kb sequence homology in 3'-terminal regions of RNA-1 and RNA-2 of a birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate.

A series of cDNA clones has been made from the birch isolate of cherry leaf roll nepovirus. Restriction enzyme analysis and sequencing showed that at the 3' end, RNA-1 and RNA-2 are identical for 1.5 kb. Also a 0.7 kb 3' end homology exists between the birch and rhubarb isolate. These sequences do not seem to code for any proteins; however, the sequence conservation points to a role in virus replication.

Base Sequence↗
PubMed1992

Determinants of substrate recognition by poliovirus 2A proteinase.

Poliovirus proteinase 2A (2Apro) is autocatalytically released from the viral polyprotein by cleavage in cis of a Tyr-Gly dipeptide at its own amino terminus, resulting in separation of the P1 structural and P2-P3 nonstructural protein precursors. A second Ty-Gly dipeptide within 3D polymerase is cleaved by 2Apro in trans, but this is not essential for viral proliferation. The mechanism which limits cleavage to only 2 of the 10 Tyr-Gly dipeptides within the poliovirus polyprotein has not been characterized. We have therefore undertaken a systematic mutational analysis of the VP1-2A site to elucidate determinants of substrate recognition by 2Apro. The P2 and P1' positions are important determinants for cis cleavage of this site, whereas a variety of substituents could be tolerated at the P2', P1, and P3 positions. The requirements for trans cleavage of this site were more stringent. We found that the 2Apro of coxsackievirus type A21 and rhinoviruses 2 and 14 have stringent requirements similar to those of poliovirus 2Apro for cleavage in trans.

Amino Acid Sequence↗
PubMed1991

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.

Amino Acid Sequence↗
PubMed1991

Synthesis and proteolytic processing of arabis mosaic nepovirus, cherry leaf roll nepovirus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate.

The genomic RNA components of three nepoviruses, arabis mosaic (ArMV), cherry leaf roll (CLRV), and strawberry latent ringspot (SLRV), were translated in rabbit reticulocyte lysate. Each component (except the RNA-2 of CLRV) directed the synthesis of proteins that corresponded in size to their theoretical coding capacity. The RNA-1 components of all three viruses were translated to yield polyproteins of Mr 250k, which were autocatalytically processed to yield up to five cleavage products. The primary products of translation of the RNA-2 components of ArMV (Mr 115k and 105k), CLRV (Mr 165k) and SLRV (Mr 99k and 96k) were polyproteins that were stable on incubation, but which underwent proteolytic processing in the presence of the corresponding RNA-1 and its translation products. These polyproteins were immunoprecipitated using antisera to appropriate virions indicating that the RNA-2 sequences encode the coat protein cistrons.

Animals↗
PubMed1991

Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein.

Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.

Base Sequence↗
PubMed1991

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.

DNA Mutational Analysis↗
PubMed1990

The nucleotide sequence of a satellite RNA associated with arabis mosaic nepovirus.

The nucleotide sequence of a satellite RNA (satRNA) associated with a lilac isolate of arabis mosaic virus (ArMV) was determined from cDNA copies. The sequence was 1104 nucleotides in length excluding the poly(A) tail, contained a long open reading frame which encodes a polypeptide of 360 amino acids, with an Mr of 39K. Nucleotide sequence comparisons revealed that the ArMV-associated satRNA shared 83% nucleotide identity with a satRNA from grapevine fanleaf nepovirus, but no extensive sequence homology was observed with other nepoviral satRNAs.

Base Composition↗
PubMed1990

Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.

Picornaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differences among them in events leading to the initiation of protein synthesis. A large segment of the 5' nontranslated region, approximately 400 nucleotides in length, promotes 'internal' entry of ribosomes independent of the non-capped 5' end of the mRNA. This segment, which we have called the internal ribosome entry site (IRES), maps approximately 200 nt down-stream from the 5' end and is highly structured. IRES elements of different picornaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, IRES elements of the genera entero- and rhinoviruses, on the one hand, and cardio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for IRES function. The IRES elements of cardio-, entero- and aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IREs is essential for translation in vitro. The IRES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown. The function of p57 or p52 in cellular metabolism is unknown. Since picornaviral IRES elements function in vivo in the absence of any viral gene products, we speculate that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation. IRES elements are useful tools in the construction of high yield expression vectors, or for tagging cellular genetic elements.

Animals↗