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. 2002 Jun;76(12):6393-7.
doi: 10.1128/jvi.76.12.6393-6397.2002.

Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles

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Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles

Ki Jeong Lee et al. J Virol. 2002 Jun.

Abstract

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral trans-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.

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Figures

FIG. 1.
FIG. 1.
(A) Effect of vTF7-3 infection on the processing of LCMV GPC. BHK-21 cells, left uninfected (lanes 1 and 3) or infected with vTF7-3 (multiplicity of infection of 3) (lanes 2 and 4), were transfected with 0.5 μg of each pT7-GP (lanes 1 and 2) or pC-GP (lanes 3 and 4) by using Lipofectamine as described previously (8, 20). Cell lysates prepared at 36 h posttransfection were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and assayed for expression of GP by a Western blot. Detection of LCMV GPC and GP-2 was done using a mouse monoclonal antibody to GP-2 (32). The positions of the GPC and GP-2 proteins are indicated on the right. The GP-2 monoclonal antibody detected a nonspecific protein in lysates of uninfected control cells (white asterisk). (B) Generation of infectious VSVΔG* pseudotype particles by complementation with LCMV GP. 293T cell monolayers in six-well plates (80% confluent) were transfected with 2 μg of the plasmid DNA indicated at the top of each panel by using Lipofectamine. Thirty-two hours after transfection, cells were infected with VSVΔG* at a multiplicity of infection of 3 PFU/cell. After a 60-min adsorption period, the inoculum was removed, cells were extensively washed with DMEM, and fresh culture medium was added. Twenty hours postinfection, supernatants were collected and clarified by low-speed centrifugation, and their infectivity was analyzed on Vero cells. Representative fields of Vero cells infected with each type of pseudotype virus are shown. Viral titers are indicated at the bottom of each panel.
FIG. 2.
FIG. 2.
Passage of CAT activity to a fresh monolayer of BHK-21 cells. 293T cell monolayers in six-well plates (80% confluent) were transfected with pC-T7 (1 μg), ARM/S-MG (0.5 μg), pC-NP (0.8 μg), pC-L (0.2 μg), pC-GP (0.5 μg), and pC-Z (0 to 0.2 μg) using the combinations indicated in the chart. Forty-eight hours posttransfection, supernatants (1.5 ml) were saved and cell lysates were prepared for CAT assay (8). Aliquots (0.5 ml) of supernatants from transfected cells were used to infect fresh monolayers of BHK-21 cells in six-well plates. After adsorption of the supernatants for 2 h, LCMV helper virus (ARM strain) was added to a multiplicity of infection of 2 PFU/cell, and adsorption was continued for another 90 min. After this, the inoculum was removed and fresh culture medium was added. Seventy-two hours postinfection, cell lysates were prepared for CAT assay. Aliquots from lysates of 293T (transfection) and BHK-21 (passage) cells were assayed for CAT activity (8). O, origin; Cm, chloramphenicol; MAc, monoacetylated chloramphenicol; DAc, diacetylated chloramphenicol.
FIG. 3.
FIG. 3.
(A) Passage of CAT activity is inhibited by treatment with anti-LCMV neutralizing antibodies but not by RNase treatment. Lanes 1 and 2 correspond to 293T cells transfected with the combination of plasmids indicated in lanes 5 and 1, respectively, of Fig. 2. Forty-eight hours posttransfection, supernatants were saved and cell lysates were prepared for CAT assay. Supernatant from cells transfected with the complete set of plasmids (lane 1) was subjected to the treatments indicated in the chart prior to being used for infection of BHK-21 cells. In all cases, except lane 8, BHK-21 cells were subsequently infected with helper LCMV at a multiplicity of infection of 2 PFU/cell. Seventy-two hours postinfection with helper LCMV, cell lysates were prepared for CAT assay. Aliquots of lysates from transfection (lanes 1 and 2) and passage (lanes 3 to 9) samples were assayed for CAT activity. (B) Monoclonal antibodies 36.1 and 197.1 to LCMV GP-1 (31) neutralized virion-associated, but not RNP-associated, LCMV infectivity. Aliquots of purified LCMV virions (200 PFU) and RNP (equivalent to 100 PFU) prepared from cytosolic extracts of LCMV-infected BHK-21 cells as described previously (8), were treated as indicated in the chart, and their infectivities were tested by standard plaque assay (virions) or transfection (RNP) under agarose overlay. Infectivity of untreated samples was considered to be 100% in each case; these values were used to normalize the infectivity of samples treated as indicated in the chart.
FIG. 4.
FIG. 4.
Expression of GFP ARM/S-MG in transfected and passage cell populations. 293T cells in six-well plates (80% confluent) were transfected with pMG-GFP (1 μg), pC-T7 (1 μg), pC-NP (0.8 μg), pC-L (0.2 μg), pC-GP (0.3 μg), and pC-Z (0.1 μg). Cells were analyzed for GFP expression 48 h after transfection (lower panels). Supernatants were collected 48 h after transfection, and aliquots (0.3 ml) were used to infect fresh monolayers of BHK-21 cells seeded on M24 plates. Cells were incubated with supernatants for 4 h before adding helper LCMV (MOI of 2 PFU/cell). After 90 min, the inoculum was removed and fresh medium was added. Sixty hours postinfection the passage culture was examined for GFP expression (top panels). GFP-positive foci (one to five per M24 well) were only detected in the passage of cells transfected with GP and Z in addition to L and NP.

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