Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.
Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.
Farley
Daniel C
DC
Oxford BioMedica Ltd., The Medawar Centre, Robert Robinson Avenue, Oxford Science Park, Oxford OX4 4GA, UK. D.Farley@oxfordbiomedica.co.uk
Iqball
Sharifah
S
Smith
Joanne C
JC
Miskin
James E
JE
Kingsman
Susan M
SM
Mitrophanous
Kyriacos A
KA
D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D013045:Species Specificity; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014764:Viral Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D014771:Virion / Q000737:chemistry
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D014759:Viral Envelope Proteins; D014764:Viral Proteins