A block to human immunodeficiency virus type 1 assembly in murine cells - PubMed (original) (raw)

A block to human immunodeficiency virus type 1 assembly in murine cells

R Mariani et al. J Virol. 2000 Apr.

Abstract

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.

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Figures

FIG. 1

FIG. 1

Derivation of MGT5 and MGT5.cyT reporter cells. (A) NIH 3T3 cells were transfected with pLTR.EGFP, which contains the HIV-1 LTR linked to the EGFP gene. hu-CD4, hu-CCR5, and hu-cyclin T1 were introduced using pBABE retroviral vectors (36). MGT5.cyT was transduced with BABE.CyT.hygro; MGT5 contains control vector BABE.hygro. (B) FACS analysis of CCR5 and CD4 on MGT5, MGT5.cyT, GHOST.R5, and NIH 3T3 cells.

FIG. 2

FIG. 2

Cyclin T1 restores HIV expression but not replication in the murine cell line. (A) Murine and human cell lines were infected with 25 ng of p24_gag_ NL4-3(VSV-G) and photographed under UV light 48 h later. Uninfected cells are shown below. (B) Murine and human cells were infected with the amounts of NL4-3(VSV-G) p24_gag_ indicated on the right. The percentage of cells within the gate defined by the vertical line in each panel is indicated. (C) MGT5, MGT5.cyT, and GHOST.R5 cells were infected with M-tropic HIV-1 isolates JR.FL and MJM at an MOI of 0.05, and p24_gag_ production was measured for 14 days. Continuation of the cultures for up to 4 weeks did not result in virus production from the murine cells (data not shown). FIG.2—Continued.

FIG. 2

FIG. 2

Cyclin T1 restores HIV expression but not replication in the murine cell line. (A) Murine and human cell lines were infected with 25 ng of p24_gag_ NL4-3(VSV-G) and photographed under UV light 48 h later. Uninfected cells are shown below. (B) Murine and human cells were infected with the amounts of NL4-3(VSV-G) p24_gag_ indicated on the right. The percentage of cells within the gate defined by the vertical line in each panel is indicated. (C) MGT5, MGT5.cyT, and GHOST.R5 cells were infected with M-tropic HIV-1 isolates JR.FL and MJM at an MOI of 0.05, and p24_gag_ production was measured for 14 days. Continuation of the cultures for up to 4 weeks did not result in virus production from the murine cells (data not shown). FIG.2—Continued.

FIG. 3

FIG. 3

Murine cells support efficient HIV-1 provirus formation. Cells were infected with NL4-3(VSV-G) at an MOI of 1. Genomic DNA was prepared 2 days later, and proviral copy numbers were determined by quantitative-competitive PCR. Competitor DNA deleted between the _Nhe_I and _Hpa_I sites flanking env was added in the amounts indicated above each lane. DNA from OM10.1 cells containing a single provirus per cell was used for calibration. The proviral load per cell calculated from band intensities normalized to OM10.1 was as follows: GHOST.R5 = 1, MGT5 = 0.5, and MGT5.cyT = 0.25.

FIG. 4

FIG. 4

Murine cells produce unspliced and spliced HIV-1 transcripts. Murine and human cells (2 × 106) were infected with NL4-3(VSV-G) at an MOI of 1. At 2 days postinfection, total RNA was prepared and HIV RNA transcripts were detected by Northern blotting using an LTR probe. Unspliced (US), singly spliced (SS), and multiply spliced (MS) transcripts are indicated by arrows. The filter was reprobed with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe to control for equal RNA loading. The ratio of unspliced to multiply spliced transcripts was 11.4 for GHOST.R5 and 5.0 for MGT5.cyT; the ratio of intensities of the unspliced transcript GHOST.R5 compared to MGT5.cyT was 3.0 and for multiply spliced it was 1.3, as determined by PhosphorImager analysis.

FIG. 5

FIG. 5

HIV-1-infected murine cells synthesize viral proteins but fail to efficiently release virions. Murine and human cells were infected with NL4-3(VSV-G) at an MOI of 1. At 3 days later, virions were pelleted from the culture medium and cell lysates were prepared. Viral proteins in the virions and cell lysates were visualized on a Western blot probed with patient serum. For the lysates, a fixed amount of protein from the murine cells and 10-fold-decreasing amounts of protein starting at 5 μg from the human cells was loaded. For the virions, amounts corresponding to 1.0 or 0.1 ml were loaded, as indicated above the lanes.

FIG. 6

FIG. 6

Infected murine cells contain Gag at levels sufficient to allow virion assembly. (A) OM10.1 cells were treated with 5.0, 1.0, 0.2, 0.02, 0.01, 0.005, or 0 U of TNF-α per ml in the lanes below the triangle. GHOST.R5 and MGT5.cyT were infected with NL4-3(VSV-G), and lysates and virions from each culture were prepared 3 days later. Viral proteins were analyzed on Western blots with AIDS patient serum. (B) p24_gag_ concentration in lysates and virions.

FIG. 7

FIG. 7

Murine cells produce infectious virus. (A) Murine and human cells were infected with NL4-3(VSV-G). At 3 days postinfection, the density of virions in the supernatant was measured by sucrose density gradient centrifugation. (B) p24_gag_ was quantitated in cell lysates and from pelleted virions. (C) The infectious titer (TCID50) of supernatant virus was measured by end-point dilution on CEM.LTR.GFP cells. The ratio of TCID50 to supernatant p24_gag_ is 154 and 236 for the MGT5.cyT- and GHOST.R5-produced viruses, respectively.

FIG. 8

FIG. 8

Ultrathin-section electron microscopic analysis of infected MGT5.cyT cells. (A) Overview of an infected MGT5.cyT cell. (B) Detail of an infected cell, revealing a complex vesicular cytoarchitecture. Note the thickened contrast-rich membranes situated in close association with cisternae of the endoplasmic reticulum (arrowheads). Adjacent is a dense body (D), also surrounded by a thickened membrane. (C to E) Budding structures and immature and mature HIV-1 particles, respectively.

FIG. 9

FIG. 9

Efficient syncytium formation but decreased coreceptor-mediated entry in the murine cell line. (A) The murine and human cells were infected with a mixture of JR.FL pseudotype containing a firefly luciferase reporter gene and VSV-G pseudotype containing Renilla luciferase reporter gene. The activities of the two luciferase enzymes were quantitated using reagents discriminating between the two enzymes. Data are reported as the ratio of JR.FL- to VSV-G-mediated infection (firefly divided by Renilla luciferase activity in counts per second). (B) Syncytium formation assay in which 293T cells expressing transfected JR.FL Env were cocultured for 18 h with MGT5 or GHOST.R5. Representative fields are shown.

FIG. 9

FIG. 9

Efficient syncytium formation but decreased coreceptor-mediated entry in the murine cell line. (A) The murine and human cells were infected with a mixture of JR.FL pseudotype containing a firefly luciferase reporter gene and VSV-G pseudotype containing Renilla luciferase reporter gene. The activities of the two luciferase enzymes were quantitated using reagents discriminating between the two enzymes. Data are reported as the ratio of JR.FL- to VSV-G-mediated infection (firefly divided by Renilla luciferase activity in counts per second). (B) Syncytium formation assay in which 293T cells expressing transfected JR.FL Env were cocultured for 18 h with MGT5 or GHOST.R5. Representative fields are shown.

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