TR1.3 viral pathogenesis and syncytium formation are linked to Env-Gag cooperation

Abstract

Infection with murine leukemia virus (MLV) TR1.3 or the related molecular construct W102G causes severe neuropathology in vivo. Infection is causally linked to the development of extensive syncytia in brain capillary endothelial cells (BCEC). These viruses also induce cell fusion of murine cell lines, such as SC-1 and NIH 3T3, which are otherwise resistant to MLV-induced syncytium formation. Although the virulence of these viruses maps within the env gene, the mechanism of fusion enhancement is not fully determined. To this end, we examined the capacity of the syncytium-inducing (SI) TR1.3 and W102G MLVs to overcome the fusion inhibitory activity inherent in the full-length Env cytoplasmic tail. These studies showed that the TR1.3 and W102G Envs did not induce premature cleavage of p2E, nor did they override p2E fusion inhibition. Indeed, in the presence of mutations that disrupt p2E function, the TR1.3 and W102G Envs significantly increased the extent of cell fusion compared to that with the non-syncytium-inducing MLV FB29. Surprisingly, we also observed that TR1.3 and W102G Envs failed to elicit syncytium formation in these in vitro assays. Coexpression of gag-pol with env restored syncytium formation, and accordingly, mutations within gag-pol were used to examine the minimal functional requirements for the SI phenotype. The results indicate that both gag-dependent particle budding and cleavage of p2E are required to activate the SI phenotype of TR1.3 and W102G viruses. Collectively, these data suggest that the TR1.3 and W102G viruses induce cell fusion by the fusion-from-without pathway.

FIG. 1.

TR1.3 does not prematurely cleave p15E. Western blot analysis of p15E and p12E was performed with cell lysates or cell-free supernatants from FB29-, TR1.3-, W102G virus-, or mock-infected cell cultures. 293T cells were cotransfected with FB29, TR1.3, or W102G Env expression plasmid and the pHIT60 Gag-Pol expression plasmid, supernatants were harvested after 48 h, equal-volume lysates or cell-free supernatants were clarified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of envelope cleavage products was detected using a pan-specific polyclonal rabbit serum directed against p15E.

FIG. 2.

Impact of p2E+ and p2E− mutations on p15E cleavage. Western blot analysis was performed to detect gp70, p15E, and p12E cleavage in the P2E+ and P2E− envelope mutants. 293T cells were cotransfected with FB29, FB29p2E+, or FB29p2E− Env expression plasmid and the pHIT60 Gag-Pol expression plasmid. Supernatants were harvested at 48 h posttransfection and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of gp70, p15E, and p12E was detected by a polyclonal antibody to either gp70 (top) or p15E (bottom).

FIG. 3.

W102G virus-mediated cell fusion is independent of p15E cleavage. (A) Cell fusion of NIH 3T3 cells following transient transfection with either wild-type, p2E+, or p2E− FB29 (dark bars), TR1.3 (striped bars), and W102G (light bars) Env expression plasmids. Results are presented as the total number (log scale) of nuclei in syncytia per field (10× objective; average of five random fields per well). Each assay was performed in triplicate, and averages for a representative assay are shown. This assay was repeated four times with similar results. (B) Cell fusion of NIH 3T3 cells following overlay with receptor-negative 293T cells transiently transfected with either wild-type, p2E+, or p2E− FB29 (dark bars), TR1.3 (striped bars), or W102G (light bars) Env expression plasmids. Each assay was performed in triplicate. This assay was repeated twice with similar findings. Asterisks indicate statistically significant differences (P < 0.05) between FB29 and TR1.3 Envs or FB29 and W102G Envs, as determined by the unpaired Student t test.

FIG. 4.

W102G mutation enhances the kinetics of cell fusion. Cell fusion of NIH 3T3 cells (red) was examined following overlay with receptor-negative 293T cells (green) transiently transfected with either FB29, TR1.3, W102G, or a vector control. The magnitude of cell fusion (yellow) over time (70 min) was determined by sequential image analysis using a Zeiss confocal microscope and LSM510META, version 3.2, image software. (A) Confocal microscope images 25 and 70 min after cell mixing. Magnification, ×10. Arrows indicate representative areas of syncytium formation. (B) Quantitative analysis of cell fusion over time. Black boxes, TR1.3; triangles, W102G mutant; circles, FB29; smooth gray line, empty plasmid. The graph represents data from three experiments performed in triplicate.

FIG. 5.

Gag-Pol coexpression is required for W102G Env-induced cell fusion. (A) Impact of wild-type Gag-Pol on Env-induced cell fusion. NIH 3T3 cells were cotransfected with either FB29 (dark bars), TR1.3 (striped bars), or W102G (light bars) Env and with wild-type Gag-Pol expression vectors. The amount of fusion was determined by counting the total number of nuclei in syncytia (five random fields; 10× objective) at 48 h posttransfection. Data shown are averages for triplicate wells. (B) Impact of altered Gag-Pol constructs on Env-induced cell fusion. The assays were performed as described for panel A, using the Gag-Pol expression vectors expressing Gag-Pol PAAA, Gag-Pol P150L, and Gag-Pol R119C/P150L. Each assay was performed in triplicate. This assay was repeated twice with similar findings. Asterisks indicate statistically significant differences (P < 0.05) between FB29 and TR1.3 Envs or FB29 and W102G Envs, as determined by the unpaired Student t test.

FIG. 6.

Formation of mature viral particles is required for W102G Env-induced cell fusion. (A) Formation of virus particles with either wild-type or altered Gag-Pol. 293T cells were cotransfected with FB29 or W102G Env and the indicated Gag-Pol construct. Levels of budding were approximated by measurement of ³²P incorporation in a reverse transcriptase assay (39). Each sample was run in triplicate, with averages shown. Asterisks indicate statistically significant differences compared to pcDNA3.1-transfected cells, as determined by the unpaired Student t test. (B) Western blot analysis of p15E and p12E in viral supernatants from 293T cells cotransfected with FB29 Env and the indicated Gag-Pol construct. Samples were prepared and analyzed as described in the legend to Fig. 1, with the exception of the PAAA sample, which was concentrated 10-fold using a microcentrifuge concentrator prior to gel loading. This blot is representative of three independent experiments.