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Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

ABSTRACT: The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

SUBMITTER: Vellinga J

PROVIDER: S-EPMC371045 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

Vellinga Jort J Rabelink Martijn J W E MJ Cramer Steve J SJ van den Wollenberg Diana J M DJ Van der Meulen Hans H Leppard Keith N KN Fallaux Frits J FJ Hoeben Rob C RC

Journal of virology 20040401 7

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amin ...[more]

PMID: 15016870

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Project description:Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.

Project description:The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Project description:Background ? Carboxyl terminus 1 (?CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that ?CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase C? phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of ?CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study ?CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase C? phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the ?CT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an ?-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that ?CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of ?CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of ?CT1 (?CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of ?CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

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Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

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Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

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