Separable mechanisms of attachment and cell uptake during retrovirus infection

Abstract

In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1) gag and pol, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.

FIG. 1

Genetic organization of plasmids used to produce replication-defective HIV-1-based retroviral vector particles. (a) Plasmid p107ΔΨΔ3′LTR, which expresses the HIV gag, pol, vif, vpr, vpu, tat, and rev genes. nef and the 3′ LTR are replaced by the rabbit β-globin polyadenylation signal (PA). (b) Vector plasmid pGFPRNL-HIV, which provides the packageable viral RNA and expresses GFP from the 5′ LTR and the neomycin phosphotransferase gene (Neo^r) from an internal RSV LTR promoter. RRE, rev-responsive element.

FIG. 2

Physicochemical properties of HIV-1 particles. (a) Sucrose density gradient analysis of native VSV-G-pseudotyped HIV-1 vector particles produced by triple transfection of 293 cells; (b) sucrose density gradient characterization of in vitro-assembled HIV-1 GPR particles with VSV-G protein. The left ordinate shows the number of G418-resistant (G418^R) colonies; the right ordinate shows levels of p24 and sucrose densities. Fractions were collected and analyzed for p24 by ELISA; infectious virus particles were assayed by infection of HT1080 cells.

FIG. 3

Binding and infectivity of HT1080 cells pretreated with VSV-G. (a) Fluorescence-activated cell sorting analysis of VSV-G vesicles bound to HT1080 cells. HT1080 cells were incubated with two different amounts of VSV-G vesicle, and binding of VSV-G to the cell surface was detected by reaction with antibody I1 to VSV-G followed by labeling with fluorescein isothiocyanate-coupled anti-mouse secondary antibody. In all cases, a total of 10,000 events were visualized. (b) Infectivity of HT1080 cells expressed as the number of G418-resistant colonies following GPR infection of HT1080 cells preexposed to 1 to 100 μg of VSV-G protein. The concentration of VSV-G is in the range of 0.5 to 50 μg/ml.

FIG. 4

A proposed model for interaction of envelope protein-free retroviral GPR particles with VSV-G vesicles and with target cells. In mechanism 1, VSV-G is complexed with GPR particles to produce infectious particles that interact with unmodified regions of the cellular membrane and allow cell uptake and infection. Alternatively, in mechanism 2, GPR particles devoid of envelope protein interact with regions of the cell membrane to which VSV-G had previously been complexed. Mechanism 3 indicates that envelope-free GPR particles also are able to bind effectively to regions of the cellular membrane not containing retroviral envelope proteins but are then able to infect ells upon subsequent addition of VSV-G. Effective binding of VSV-G alone to the cellular lipid membrane is indicated in mechanism 4.