The cellular protein La functions in enhancement of virus release through lipid rafts facilitated by murine leukemia virus glycosylated Gag

Abstract

Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65(gag) for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80(gag)). gPr80(gag) is translated from the same unspliced viral RNA as Pr65(gag), from an upstream in-frame CUG initiation codon. As a result, gPr80(gag) contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80(gag) into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80(gag) facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80(gag) is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80(gag) function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80(gag). Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La.

FIG 1

Expression plasmids used. Diagrams of the M-MuLV expression plasmids used in these experiments are shown. AKAQ188 has deletions in the region encoding the leader peptide (deletion of positions 215 to 561), containing both the packaging signal (positions 217 to 567) (46) and the CUG initiation codon for gPr80^Gag (positions 357 to 359), and in the env gene (deletion of positions 5819 to 7197). The CUG start codon for gPr80^gag was replaced by an AUG start codon in both p8065-2 and HA-gg88. In HA-gg88, viral sequences upstream of the CUG codon were also removed so that this plasmid would lack a functional packaging sequence (46). LTR, long terminal repeat; CMV-P, cytomegalovirus promotor.

FIG 2

The 88 amino acids at the amino terminus in gPr80^Gag facilitates M-MuLV particle release through lipid rafts. The M-MuLV Gag-Pol expression vector was transfected with p8065-2 and HA-gg88 into 293T cells. (A) The cells and media were harvested at 48 h posttransfection, and the same portion of cells and viruses were subjected to the Western blots with anti-p30^CA and anti-β-Tubulin (loading control). (B) The cell lysates prepared from the transfected 293T cells with the lysis buffer containing 1% Triton X-100 were analyzed by membrane floatation in 5 to 30% sucrose gradients. Low-density fractions represented detergent-resistant membranes (DRM) and lipid rafts, while high-density fractions represented protein in detergent-soluble membranes (DSM) or cytosolic proteins. (C) The amount of Pr65^gag was quantified by densitometry, and the relative amount of Pr65 in lipid rafts to control are shown (means ± standard deviations [SD]). (D) Viruses harvested from the transfected 293T cells were analyzed by density gradient centrifugation, and distribution of p30^CA was analyzed by Western blotting and densitometry.

FIG 3

Localization of HA-gg88, NA14, and Ro. The 293T cells were transfected with HA-gg88. The transfected cells were fixed at 36 to 48 h posttransfection with paraformaldehyde and then incubated with anti-HA and anti-NA14 antibodies. The antigens and nuclei were visualized by secondary antibodies conjugated with Alexa 488, Alexa 546, and DAPI (4′,6-diamidino-2-phenylindole). The panels for NA14-DAPI and HA-DAPI and a merge of the first two panels are shown. The inserts are enlarged images and are shown next to the merge picture.

FIG 4

Relocalization of La by HA-gg88. The 293T and NIH 3T3 cells were transfected with HA-gg88 and the JSRV Env-expressing vector ΔGP-HA (transfection control). The transfected cells were fixed at 36 to 48 h posttransfection with paraformaldehyde and then incubated with anti-HA and anti-La antibodies. The antigens were visualized by the secondary antibodies conjugated with Alexa 488 and Alexa 546. The panels for La, HA, and a merge (La-HA-DAPI) are shown.

FIG 5

Overexpression of La facilitates M-MuLV particle release through lipid rafts. The M-MuLV gag-Pol expression vector (AKAQ188) was transfected with mouse La and human La expression vectors into 293T cells. The assessment for viral release (A), the distribution of Pr65^gag in DRM (B and C), and the density of released viruses from the transfected cells (D) are described in the legend to Fig. 2.

FIG 6

Interference of La expression antagonizes HA-gg88 in M-MuLV particle release. M-MuLV Gag-Pol expression vector is transfected into 293T cells with or without HA-gg88. At 24 hours posttransfection, siRNAs against human La (no. 5 and no. 10) were transfected. Two days after siRNA transfection, media were replaced, and then the cells and media were harvested after 8 h of further incubation. The same portion of cells and viruses were subjected to the Western blots with anti-p30^CA, anti-La, and anti-β-Tubulin (loading control). NSP, non-specific signals.

FIG 7

Effect of IFN-α on virus release from 293T cells overexpressing HA-gg88 and mouse La. The 293T cells were transiently transfected with M-MuLV Gag-Pol expression vector with HA-gg88 and a mouse La expression vector. The cells were treated with different concentrations of IFN-α for 24 h, after which media were replaced, and the cells and released viruses were collected after 6 h of further incubation. The same portions of cells and viruses were subjected to the Western blot analyses with anti-p30^CA.

FIG 8

Overexpression of HA-gg88 and La increases the efficiency of HIV-1 particle release. The HIV-1 Gag-Pol expression vector was transfected into 293T cells with p8065-2 and HA-gg88 (A) or mouse La and human La expression vectors (B). The cells and media were harvested at 48 h posttransfection, and the same portion of cells and viruses were analyzed by Western blotting with anti-HIV p24^CΑ and anti-β-Tubulin (loading control).