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. 1998 Aug;72(8):6527-36.
doi: 10.1128/JVI.72.8.6527-6536.1998.

Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors

E Poeschla  1 , J Gilbert, X Li, S Huang, A Ho, F Wong-Staal
Affiliations

Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors

E Poeschla et al. J Virol. 1998 Aug.

Abstract

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5' splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34(+) CD38(-) human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.

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Figures

FIG. 1
FIG. 1
Schematic representation of the HIV-2 lentivirus vector system. (A) Nucleotides deleted from the ψ region of HIV-2KR.; (B) the protein expression plasmid used for trans packaging and the VSV-G expression plasmid supplying the deleted env function; (C) HIV-2-based vectors. Abbreviations: BGH, bovine growth hormone; CMV, cytomegalovirus.
FIG. 2
FIG. 2
ψ deletion alone abrogates replication but preserves transient HIV-2 protein expression. HIV-2KR proviral plasmids pE32Δψ and pE40 were transfected into Molt4-8 T cells. The ordinate cutoff is 10 pg of p26/ml, the limit of sensitivity of the assay.
FIG. 3
FIG. 3
RNase protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 106 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free DNase I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
FIG. 4
FIG. 4
p26 antigen production at 48 h in supernatants of COS-1 cells electroporated with HIV-2 expression plasmids. Bars indicate standard errors.
FIG. 5
FIG. 5
Effect of mitotic arrest on an HIV-2 lentivirus vector compared to an Mo-MuLV retrovirus vector. HeLa cells were arrested in the G1/S phase by treatment with 20 μg of aphidicolin (aphid.) per ml; cell cycle arrest (<0.1% G2/M) was verified by flow cytometry after propidium iodide staining.
FIG. 6
FIG. 6
Assay for transfer of HIV-2 coding sequences. Nested PCR amplifications followed by Southern blotting with an internal 32P-labeled pol probe were performed with 1 μg of genomic DNA (present in all tubes except PCR blank in lane 1) from HeLa cells transduced at a high MOI, yielding an efficiency of >98% as described in the text. Lanes: 1, PCR without genomic DNA; 2 to 5, reactions containing 1 μg of genomic DNA from transduced cells; 6 to 12, reactions containing 1 μg of the same DNA from the L15.7-transduced cells coamplified with various cell equivalents of genomic DNA prepared from HIV-2-infected T cells: lane 6, 1 cell; lane 7, 5 cells; lane 8, 50 cells; lane 9, 100 cells; lane 10, 500 cells; lane 12, 1,000 cells.
FIG. 7
FIG. 7
Flow cytometric analysis for GFP expression in a T-cell line (Molt4; A) and a monocytoid cell line (U937; B) 48 h after transduction with an HIV-2 GFP vector (MOI = 1.0). Solid lines, untransduced control cells; dashed lines, transduced cells. Fl., fluorescence.
FIG. 8
FIG. 8
PCR analyses of transduced CD34+ cell subsets for lacZ and β-globin DNA (single-copy cellular gene DNA input control). Cells were transduced with DNase-treated vectors and sorted by flow cytometry into CD34+ CD38− and PKH26 hi or PKH26lo fractions before DNA extraction, PCR, and Southern blotting with an internal lacZ or β-globin probe. (A) Analysis of cells FACS sorted as CD34+ CD38− and PKH26hi or PKH26lo after transduction in the presence (+) or absence (−) of cytokines as detailed in Materials and Methods. (B) Cells were transduced with each DNase-treated vector in the presence of cytokines and similarly sorted. LacZ standards (lanes a to h): 0, 1, 4, 16, 64, 256, 1,024, and 4,096 copies of the lacZ gene; β-globin standards (lanes a to f): genomic DNA equivalent to 0, 1, 5, 50, 500, and 5,000 U937 cells. β-globin PCRs for both experiments were amplified simultaneously; the standard curve is shown in the bottom panel of B. P, PCR blank; M, DNase-treated Mo-MuLV (LZRNL) vector supernatant; H, DNase-treated HIV-2 lacZ vector supernatant.
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