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. 2006 Apr;80(8):4191-5.
doi: 10.1128/JVI.80.8.4191-4195.2006.

Cellular factors required for Lassa virus budding

Shuzo Urata  1 , Takeshi Noda, Yoshihiro Kawaoka, Hideyoshi Yokosawa, Jiro Yasuda
Affiliations

Cellular factors required for Lassa virus budding

Shuzo Urata et al. J Virol. 2006 Apr.

Abstract

It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicular body pathway functionally. Our data may provide a clue to develop an effective antiviral strategy for Lassa virus.

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Figures

FIG. 1.
FIG. 1.
Effects of dominant-negative or wild-type host cell factors. (A) COS-7 cells were cotransfected with pCLV-Z and the empty vector as a control (Control) or with the expression plasmid for AP3δ-5′, Nedd4-WW, Vps4AEQ, Vps4BEQ, or AIPΔN. Extracellular VLPs were pelleted from the culture fluids. VLP-associated or cell-associated Z was detected by Western blotting (WB) using rabbit anti-Z antiserum. WB using anti-Flag monoclonal antibody was also used to examine the expression of Flag-tagged AP3δ-5′, Nedd4-WW, Vps4AEQ, Vps4BEQ, or AIPΔN in cells (top). (B and C) COS-7 cells were cotransfected with pCLV-Z and the empty vector (Control) or the expression plasmid for Vps4A, Vps4B, AIP1, or Tsg101. VLP-associated or cell-associated Z and FLAG-tagged proteins were detected as described for panel A. The expression of Myc-tagged Tsg101 was examined by WB using an anti-Myc monoclonal antibody. On the right are shown the intensities of the bands for cell and VLP-associated Z (left) that were quantified using a LAS3000 imaging system (Fuji Film). The efficiency of Z-induced VLP budding in cells cotransfected with pCLV-Z and the control vector (VLP/cellular) was set to 1.0. The data are shown as averages and standard deviations of three independent experiments.
FIG. 2.
FIG. 2.
Lassa virus-like particles produced by expression of Z. At 24 h posttransfection of COS-7 cells (B and C) or 293T cells (D) with pCLV-Z, pleomorphic particles were seen budding from the plasma membrane. (A) Control COS-7 cells. EM was performed as described previously (16). Bars, 300 nm.
FIG. 3.
FIG. 3.
Effects of siRNA specific for Tsg101, AP3δ, Nedd4, Vps4A, or AIP1 on Lassa VLP budding. (A, C, D, E, and F) 293T cells were pretreated with siRNA specific for Tsg101 (siTsg101), AP3δ (siAP3δ), Nedd4 (siNedd4), Vps4A (siVps4A), or AIP1 (siAIP1) or with control RNA (Control) 1 day before plasmid transfection. The following day, these cells were cotransfected with pCLV-Z and siRNA or control RNA. Endogenous Tsg101, AP3δ, or Nedd4 was detected by mouse anti-Tsg101 (A, top), anti-adaptin monoclonal antibody (C, top), or rabbit anti-Nedd4 polyclonal antibody (D, top), respectively. FLAG-tagged Vps4A or hemagglutinin (HA)-tagged AIP1 was detected by mouse anti-FLAG (E, top) or anti-HA (F, top) monoclonal antibody, respectively. Cell-associated (middle) and VLP-associated (bottom) Z proteins were detected by Western blotting (WB) using rabbit anti-Z antiserum. (B) The intensity of the band for VLP-associated Z in panel A was quantified as described in the legend to Fig. 1. The extent of Z in the VLPs released from cells cotransfected with pCLV-Z and the control siRNA was set to 1.0. The data are shown as averages and standard deviations of three independent experiments.
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