Oncolytic virotherapy is an emerging treatment option for numerous cancers, with several virus families currently being evaluated in clinical trials. More specifically, vaccine-strain measles virus has arisen as a promising candidate for the treatment of different tumour types in several early clinical trials. Replicating viruses, and especially RNA viruses without proofreading polymerases, can rapidly adapt to varying environments by selecting quasispecies with advantageous genetic mutations. Subsequently, these genetic alterations could potentially weaken the safety profile of virotherapy. In this study, we demonstrate that, following an extended period of virus replication in producer or cancer cell lines, the quasispecies consensus sequence of vaccine strain-derived measles virus accrues a remarkably small number of mutations throughout the nonsegmented negative-stranded RNA genome. Interestingly, we detected a nonrandom distribution of genetic alterations within the genome, with an overall decreasing frequency of mutations from the 3\' genome start to its 5\' end. Comparing the serially passaged viruses to the parental virus on producer cells, we found that the acquired consensus mutations did not drastically change viral replication kinetics or cytolytic potency. Collectively, our data corroborate the genomic stability and excellent safety profile of oncolytic measles virus, thus supporting its continued development and clinical translation as a promising viro-immunotherapeutic.

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According to their cellular receptor use, measles virus (MV) strains can be separated into two phenotypes, CD46-using and CD46-non-using. A long chimeric receptor, CD46CD[55-46], was generated from the CD46 backbone, encompassing the four short consensus repeat (SCR) domains of CD46 linked via a flexible glycine hinge to SCR1 and SCR2 of CD55, SCR3 and SCR4 of CD46 and the STP, transmembrane and cytoplasmic tail of CD46. This chimeric receptor was proficient for MV binding but deficient in mediating MV-induced cell-to-cell fusion and virus replication, possibly due to the extended distance between the MV haemagglutinin (H) binding site (CD46 SCR1-SCR2) and the cell membrane. When coexpressed with either wild-type CD46 or CD150, this fusion-incompetent receptor exerted a dominant negative effect and inhibited both cell-to-cell fusion and entry of MV with CD46-using, but not CD46-non-using, phenotype. A soluble octameric CD46-C4bpalpha exhibited similar CD46- and CD150-mediated fusion inhibition properties only against CD46-using MV. This suggests that the long CD46CD[55-46] receptor acts by sequestering incoming MV prior to its binding to the shorter functional CD46 or CD150 receptor.

To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I-II-III-IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.

暂无摘要。

The binding of a recombinant soluble form of the measles virus (MV) hemagglutinin (sH) to cells expressing hybrid CD46/CD4 proteins was compared to that of purified virus. For binding of both ligands, both CD46 external short consensus repeats I and II (SCR I and II) in the natural order were essential. The addition of SCR III and IV enhanced virus binding but inhibited sH binding. Accordingly, this lowered the ability of sH to compete with MV binding. Antihemagglutinin monoclonal antibodies selectively inhibited the binding of either sH or MV. Thus, sH and MV share a common binding site in SCR I and II but differ in their apparent avidity to CD46 under the influence of SCR III and IV.

暂无摘要。