Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry

Abstract

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.

FIG. 1

Construction of the KΔUs3-8Z recombinant virus and structure of the VSV-G/HSV chimeric proteins. (A) Schematic of the HSV-1 genome depicting the location of the essential and nonessential glycoprotein genes and replacement of the Us3-Us8 BamHI J fragment of strain KOS by a lacZ expression cassette to generate the gD-null virus KΔUs3-8Z. The accompanying Southern blots demonstrate the presence in KΔUs3-8Z of the 4.2-kb lacZ cassette (LacZ probe) and the absence of the 6.5-kb BamHI J fragment (BamHI J probe). (B) Wild-type and chimeric proteins (kindly provided by Hara P. Ghosh, McMaster University) represented as boxes corresponding to the EC (left), TM (center), and CT (right), with the relevant amino acid numbers provided in each case. VSV-G sequences are shown as open boxes, HSV-1 gB sequences are shown as black boxes, and HSV-1 gD sequences are shown as grey boxes. A deletion in the gB TM creating the gB₃ derivative is indicated.

FIG. 2

Requirements for incorporation into mature HSV-1 particles. 293T cells were transfected with the plasmids indicated above each panel and were infected with the gD-null virus KΔUs3-8Z. Metabolically labeled proteins in cell lysates and extracellular virus immunoprecipitated with antibodies (Ab) against gB (lanes B), gC (lanes C), gD (lanes D), or VSV (lanes G) were separated by SDS-PAGE and visualized by autoradiography. ∗ and #, wild-type VSV-G and VSV-G/HSV chimeric proteins, respectively.

FIG. 3

Complementation of gD functions in HSV-1 entry and neutralization of complementing virus. (A) Complementation measured as PFU on VD60 cells from the media of transfected, infected 293T cells. (B) Neutralization assay results for VD60 cells from the media of transfected, infected 293T cells. Results are expressed as percent reduction in the number of plaques (e.g., “100%” indicates complete neutralization).