In Vitro Cell-Free Conversion of Noninfectious Moloney Retrovirus Particles to an Infectious Form by the Addition of the Vesicular Stomatitis Virus Surrogate Envelope G Protein

Abstract

In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV)gag and pol genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA (gag-pol RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.

Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes

“…E2 present within the viral pellets migrated slightly slower than the cell-associated forms due to modifications of the associated glycans by Golgi enzymes (not depicted). The presence of VSV-G in viral pellets generated with MLV-G2A assembly-defective core proteins is due to empty vesicles formed by VSV-G itself (48). (C) Immunoblots of lysates of 293T producer cells and of purified pseudo-particles generated with E1 or E2 expressed alone, from two separate expression units, or coexpressed in trans or in cis, from a ⌬CE1E2 polyprotein (E1E2).…”

Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12)(q22;p12)

“…The pCMV-G containing vesicular stomatitis virus envelope glycoprotein was cotransfected with pB-TS, pB-ST, and pB-T⌬ PS or vector alone as a control into 293GP cells, a retrovirus packaging cell line expressing MMLV gag and pol genes. 33,34 The IL-3-dependent murine leukemia BaF3 cells were infected with these supernatants in the presence of 5 g/mL polybrene. G418-resistant BaF3 expressing the indicated proteins and control cells were grown in RPMI 1640 supplemented with 10% FCS and 5 ng/mL recombinant murine IL-3.…”

Rescue of retroviral envelope fusion deficiencies by cationic liposomes

“…Gag‐pol expression in the presence of a suitable packagable RNA enables the production of particles identical in structure and density to infectious virus 9, with the exception that they lack any specific viral envelope proteins and so are non‐infectious. Nevertheless, infectivity can be obtained in certain situations when another means of inducing membrane fusion is provided, such as by cationic liposomes 9–11 or the fusogenic envelope protein of vesicular stomatitis virus 12. Co‐expression of a retroviral envelope protein leads to its efficient incorporation in the lipid membrane of the gag‐based particle 13.…”