Table 1.
Interaction of adaptor proteins with the RSV matrix protein in a yeast two-hybrid system.
Fig 1.
The M protein is seen associating with the AP-3Mu3A protein in distinct juxtanuclear cytoplasmic regions at 12 hours p.i. and in cytoplasmic inclusions at 24 hours p.i.
HRSV-infected and mock-infected HEp2 cells were fixed 12 h and 24h after infection and were double stained with various antibody combinations, followed by CLSM analysis. The antibodies used are as indicated: goat anti-AP-3Mu3A (1:50), mouse anti-Matrix (1:100), rabbit anti-goat Alexa-Fluor 488 (1:200), donkey anti-mouse Alexa-Fluor 546 (1:200). Primary antibodies were incubated for 60 minutes; cells were then washed with Tween 20 wash solution and then subsequently incubated for 60 minutes with secondary antibodies. DAPI, is the 405 nm output corresponding to a blue color in the image above; AP-3Mu3A, is the 488nm output corresponding to the green color in the image above; HRSV Matrix, is the 546nm output corresponding to the red color in the image above; Merge, is the computer-generated merged image of all three outputs, with yellow coloration indicating colocalization. The results were reproducible in at least three independent assays. Scale bar is 10μm.
Fig 2.
The HRSV M protein co-immunoprecipitates with the AP-3Mu3A and AP-3delta complex during HRSV infection.
HEp2 cells at approximately 90% confluency were either infected at an MOI of 5 or mock infected for 24 hours, cells were scraped or proteins were subsequently extracted using MPER. Cell lysates were incubated for 6 hours with 1 μg of either polyclonal goat anti-AP-3Mu3A or monoclonal mouse anti-AP-3delta along with a antibody specific isotype control, polyclonal goat-anti H1N1or monoclonal mouse anti-HIV-1 p24 Gag at 4°C on a rotating device. 20μl Protein A/G agarose beads were added to lysate plus corresponding antibody and incubated overnight. Immunoprecipate complex was pelleted and washed with PBS and then ran out on a SDS-PAGE gel and transferred to nitrocellulose membrane. Membrane was blocked and then probed with either monoclonal mouse anti-Matrix or polyclonal goat-anti HRSV primary antibody as described previously for one hour. Membranes were then washed with a PBS-Tween20 solution extensively and then probed with species-specific secondary antibodies donkey anti-goat IR dye 800 and donkey anti-mouse IR dye 700. Membranes were again washed extensively and blots were imaged on Odyssey Infrared imager. The results were reproducible in at least two independent assays.
Fig 3.
RSV matrix sequence alignment.
ClustalW sequence alignment of Matrix protein comparing human RSV with various animal respiratory syncytial virus strains. The available sequences are aligned via ClustalW with color coding to indicate conservation (Black = identical, pink = strong similarity, green = weak similarity, white = no similarity). The conserved YXXL among human RSV is shown in a box.
Fig 4.
Effect of mutation in YXXL domain of HRSV M protein.
A) Sequence alignment of Matrix protein amino acids 197–200 comparing various animal respiratory syncytial virus strains. The black regions highlight the conserved tyrosine and the Tyr+4 hydrophobic amino acid residue (leucine) at residues 197 and 200 respectively. [Human HRSV Long Strain (HHRSV Long), HHRSV Strain A,B (HHRSV A,B), Bovine HRSV (BHRSV), Ovine HRSV (OHRSV), Pneumonia Virus of Mice (PVM), Avian Metapneumovirus Strain A,B,C (APV A/B/C), Human Metapneumovirus Strain A (HMPV A)]. B) Schematic diagram of the Opt.M and point-mutated Opt.M constructs. The diagram highlights the proposed adaptor protein (AP) basolateral sorting motif (shown in single letter amino acid code), the putative nuclear export signal (NES) (8) and the nuclear localization signal within amino acids 110–183, the binding site for Importin beta1 (10). Single letter code values refer to the beginning of the M amino acid sequence. Bold letters indicate the putative 197-YXXL-200 and possible di-leucine sequence motif located in M from amino acids 200–202. C) The mutation of the tyrosine at amino acid residue 197 to alanine and leucine at amino acid residue 200 to alanine causes a distinct phenotypic change in the localization of the HRSV Matrix protein. HEp2 cells were transfected with indicated pEGFP-C1 Opt. M constructs and were fixed with paraformaldehyde at 24 hours post transfection as described previously. Cells were imaged for Opt.M, adaptor protein and DAPI using CLSM protocols described previously. Negative controls include transfection of cells with empty plasmid vector (pEGFP-C1) without M insert (left side panel) and mock transfected cells with staining for adaptor protein only with goat anti Ap3u3A followed by rabbit anti goat Alexa Fluor 546 antibody (right side panel). The first 3 images on the left side panel shows staining for Opt.M and DAPI. The first 3 images on the right side panel shows merged image with staining for Opt.M, adaptor protein and DAPI. The results were reproducible in at least two independent assays. D) Mutation in YXXL domain of M reduces HRSV titer. The HEp-2 cells were transfected with control plasmid, WT-M, Y197A or L200A mutant plasmids and infected with HRSV. Four days after infection, supernatants were collected, and the virus titer was measured by plaque assay. Representative data from three independent experiments are shown. The results were reproducible in at least two independent assays.
Fig 5.
AP3Mu3A is up-regulated at 24 hours post-infection in infected cells versus mock infected HEp2 cells while at 6 hours there was no statistically significant difference.
The AP3delta subunit was also analyzed and it was determined that there was no statistical significant difference in infected cells versus mock infected cells at both 6 and 24hours post-infection. 15μg of cell lysate, extracted by MPER (Pierce) were analyzed by 4–12% Bis-Tris SDS-PAGE gels (Invitrogen) in triplicate and transferred to nitrocellulose. Blots were blocked overnight in Rockland IR blocking buffer, then probed with the following antibodies diluted in 50% Tween-20 Wash Buffer: 50% Rockland blocking buffer for one hour incubation periods with extensive wash periods between incubations: Primary antibodies used are Goat anti-Ap3m1 (1:200), Mouse anti-RSV Matrix (1:1000), Rabbit anti-delta adaptin (SA4) (1:1000), Rabbit anti-GAPDH (1:10000), and secondary antibody used are Donkey anti-Goat IR 800 (1:25000), Donkey anti-Rabbit IR 800 (1:25000), Donkey anti-Rabbit IR 700 (1:5000), Donkey anti-Mouse IR 700 (1:5000), Donkey anti-Mouse IR 800 (1:20000). Blots were analyzed on Licor Imager and integral intensities were measured for each band corresponding to the 41kDa GAPDH, 47 kDa AP3Mu3A, and 160 kDa delta-adaptin bands. The statistical analysis was performed on the ratio of the integral intensity measurement of the triplicate average of the protein of interest versus the GAPDH control in infected cells versus mock-infected cells at various time points post RSV infection.
Fig 6.
Trafficking route of HRSV M and AP-3 in epithelial cells (adapted with permission from Rodriguez-Boulan et al Nature Reviews 2005, 233–247).
HRSV M protein after exiting the trans-Golgi network (TGN) interacts with AP3 adaptor protein via YXXL domain and reach common recycling endosomes (CREs) and apical recycling endosomes (AREs). The ARE serves as a slow recycling endosome as well as the final destination for basolateral to apical membrane transcytosing proteins. M may also interact with Exocyst Complex Component 6 (EXOC6), which is involved in vesicular trafficking from the Golgi to plasma membrane. The M protein then interacts with the major ARE-associated protein, Rab11 family interacting protein 2 (FIP2) and help in the budding of the virus through lipid rafts. Abbreviations: basal sorting endosomes (BSEs), late endosomes (LEs) and lysosomes (LYS), apical sorting endosomes (ASEs), common recycling endosomes (CREs), apical recycling endosomes (AREs), trans-Golgi network (TGN), Exocyst Complex Component 6 (EXOC6).