Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency

Hum Gene Ther. 2000 Jul 1;11(10):1439-51. doi: 10.1089/10430340050057512.

Abstract

A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Blotting, Western
  • Cell Line
  • Culture Media
  • Dose-Response Relationship, Drug
  • Gene Transfer Techniques*
  • Genes, env / genetics*
  • Genes, gag / genetics
  • Genes, pol / genetics
  • Humans
  • Moloney murine leukemia virus / genetics*
  • Phosphates / pharmacology
  • RNA, Messenger / metabolism
  • Rats
  • Receptors, Virus / metabolism
  • Retroviridae / genetics
  • Transduction, Genetic*
  • Tumor Cells, Cultured

Substances

  • Antibodies, Monoclonal
  • Culture Media
  • Phosphates
  • RNA, Messenger
  • Receptors, Virus
  • leukemia virus receptor, gibbon ape