Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly - PubMed (original) (raw)

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly

R Mariani et al. J Virol. 2001 Apr.

Abstract

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

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Figures

FIG. 1

Processing ability of various rodent cell lines. M. musculus (MGT5.CyT, EL4.CyT, and L1-2.CyT), M. dunni (MDTF.CyT), hamster (CHO.CyT), or rat (Rat2.CD4.R5) cell lines with or without transfected hu-cyclin T1 were infected with NL4-3(VSV-G) at an MOI of 1. (A) Immunoblot analysis of infected cell lysates and virions pelleted from supernatant with anti-HIV serum. (B) Virion production of the infected cell lines. (C) Infectious titer of virus produced by the infected cell lines. Results are the average of triplicates. (D) Calculated ratio of TCID50 to supernatant p24_gag_ from panels B and C.

FIG. 1

FIG. 2

Electron microscopy of HIV-1 infected CHO and MDTF derivatives. (Upper panels) (a) Ultrathin sections were processed by glutaraldehyde-osmium fixation and ERL embedding. Budding structures (a) and extracellular HIV-1 particles are present. (b and c) Immunoelectron microscopy with anti-CA antibody of HIV-1-infected CHO derivative shows labeled budding structures (b) and adjacent extracellular particles (c). Gold particles are scarce in the cytoplasm and nearly absent in the extracellular space. (d and e) Gold-labeled HIV-like particles in the ER cisternae. Some cytoplasmic vacuoles (V) were labeled (arrow), while adjacent vacuoles remained unlabeled (arrowhead). (Lower panels) (a) Ultrathin sections of infected, fixed, and embedded MDTF.CD4.R5.CyT cells at low magnification showed virus particles at the plasma membrane. Morphological alterations were not observed. (b) Higher magnification revealed mature extracellular HIV-1 particles adjacent to the plasma membrane. (c) The extracellular particles were immunolabeled.

FIG. 3

CHO.CD4.R5.CyT but not other rodent cells support low-level HIV-1 replication. (A) MGT5.CyT, CHO.CD4.R5.CyT, MDTF.CD4.R5.CyT, LT4.R5.CyT, and HOS.CD4.R5 cells were infected with HXB.BaL and CHO.CD4.R5.CyT and HOS.CD4.R5 cells were also infected with NL-BaL at an MOI of 0.05, and p24_gag_ production was measured over time. (B) CHO.CD4.R5.CyT and HOS.CD4.R5 were infected with HIV.YU2.GFP at an MOI of 0.05, and the percentage of green cells was scored by fluorescence-activated cell sorting analysis.

FIG. 3

FIG. 4

CHO cells are competent for coreceptor function but fail to support efficient virus replication. CHO.CD4.R5.CyT and HOS.CD4.R5 cells were infected with JR.FL pseudotyped firefly luciferase or VSV-G pseudotyped Renilla luciferase single-cycle reporter viruses. Luciferase activity was measured in cell lysates 3 days postinfection. The results are the averages of triplicates and are representative of three experiments.

FIG. 5

The assembly defect in 3T3 cells is complemented by fusion to human cells. (A) Strategy for testing HIV assembly and production in mouse-human heterokaryons. MGT5.CyT, GHOST.R5, and MDTF.CD4.R5.CyT cells were infected at an MOI of 1 with NL4-3(VSV-G), washed three times, and fused 3 days later to 293T, 3T3, or MDTF cells. After 24 h postfusion, the cells were lysed for immunoblot analysis, and virions were collected for p24_gag_ and TCID50 measurement. To control for input virus contamination, mock fusions in which PEG was omitted were analyzed in parallel. (B) p24_gag_ concentration in heterokaryon supernatants. (C) TCID50 measurement of virus in heterokaryon supernatants. (D) TCID50 measurement of virus in _M. dunni_-human heterokaryon supernatants. (E) Immunoblot analysis of heterokaryon lysates using anti-HIV serum.

FIG. 6

Fusing human cells to infected murine cells that express hu-cyclin T1 increases LTR transactivation. Murine or human cells were infected with luciferase reporter virus. Three days postinfection, the cells were cocultivated with JR.FL Env-expressing or control cells for an additional 24 or 48 h to allow Env-mediated fusion between the two cell types. Luciferase activity in the cell lysates was then measured. Results are the averages of triplicates and are representative of three experiments.

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Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly - PubMed (original) (raw)