Sequences in the UL11 tegument protein of herpes simplex virus that control association with detergent-resistant membranes

Abstract

The product of the UL11 gene of HSV-1 is a small, membrane-bound tegument protein with features that are conserved among all herpesviruses. For all viruses examined, mutants lacking this protein (or its homolog) have budding defects and accumulate capsids in the cytoplasm of the infected cell. UL11 binds to the cytoplasmic faces of host membranes via N-terminal myristate and nearby palmitate moieties. These fatty-acid modifications are typical of proteins that localize to detergent-resistant membranes (DRMs), and the experiments described here revealed that a small amount (approximately 10%) of UL11 retains the ability to float in sucrose gradients following treatment of cells with Triton X-100. However, mutants lacking sequences previously shown to be involved in the trafficking of UL11 from the plasma membrane (LI and acidic cluster motifs) were found to have a dramatically increased association with DRMs. These findings emphasize the dynamic properties of this poorly-understood but conserved tegument protein.

FIG. 1

Flotation analysis of UL11. (A) A7 cells transfected with a UL11-GFP expression vector were metabolically labeled for 2.5 h and osmotically disrupted, as described in the text. Cytoplasmic membranes were treated with nothing (TM, total membranes), 0.5% TX-100 (DRM, detergent-resistant membranes), or 0.5% SDS (negative control). The ability of UL11-GFP to float to the upper fractions of sucrose step gradients during centrifugation was examined, and representative autoradiograms, obtained following immunoprecipitation and SDS-PAGE analysis, are shown. As a control for DRM disruption, endogenous transferrin receptor (TfR) was monitored in one experiment following radiolabeling, TX-100 treatment, and flotation. The tops and bottoms of the gradients are indicated. (B) Phosphorimager analysis was used to quantitate the flotation results, which are shown as the percentage of floating protein (top three fractions) relative to the total protein (all fractions). The averages of four experiments are shown, along with the standard deviations. (C) Flotation assays were used to compare the membrane-binding properties of radiolabeled UL11-GFP produced in transfected cells with untagged UL11 and UL11-GFP produced by wild-type and recombinant viruses (HSV and HSV.GFP, respectively). Cells were labeled for the final 2.5 h of infection, harvested, and floated as described in the text. The averages from at least four experiments are shown, along with the standard deviations. (D) To examine the saturability of DRMs, cells were transfected with increasing amounts of plasmid DNA, metabolically labeled, and subsequently analyzed for UL11-GFP expression levels (hatched bars; RPU = relative phosphorimager units) and DRM localization (grey bars). A repeat of this experiment gave comparable results.

FIG. 2

DRM association of UL11 mutants. (A) Diagram of UL11-GFP and the mutants that were analyzed. The motifs of interest are shown: G, myristylation site; CCC, palmitylation site; LI, di-leucine-like; DIESEEE, acidic cluster (AC). Sites of alanine substitutions are indicated. (B) To examine the reactivity of the mutants to anti-UL11 and anti-GFP sera, transfected cells were metabolically labeled and immunoprecipitated proteins were analyzed by SDS-PAGE. The positions of the UL11-GFP species are indicated with a bracket to the left. Positions of markers (in kDa) are indicated to the right. (C) Constructs depicted in panel A were analyzed for their ability to float in the absence (TM) and presence (DRM) of 0.5% TX-100. Each construct was analyzed a minimum of three times.

FIG. 3

Analyses of N-terminal UL11 chimeras. N-terminal extensions corresponding to the first 10 amino acids of (A) Src or (B) Fyn were attached to UL11-GFP, as represented by the shaded boxes in the diagrams. Wavey lines denote fatty acid modifications and “+” indicates basic residues. These N-terminal chimeras and the indicated mutants (defined in Fig 2A) were analyzed for their ability to float in the absence (TM) and presence (DRM) of 0.5% TX-100. Each construct was anlayzed a minimum of three times