ORIGINAL ARTICLE

A critical role of LAMP-1 in avian reovirus P10 degradationassociated with inhibition of apoptosis and virus release

Haiyang Wu1,2,3 • Zhiyuan He1,2,3 • Jun Tang1,2,3 • Xiaoqi Li1,2,3 • Hong Cao1,2,3 •

Yongqiang Wang1,2,3 • Shijun J. Zheng1,2,3

Received: 6 September 2015 / Accepted: 17 December 2015 / Published online: 7 January 2016

� Springer-Verlag Wien 2016

Abstract Avian reovirus (ARV) causes viral arthritis,

chronic respiratory diseases, retarded growth and malab-

sorption syndrome. The ARV p10 protein, a viroporin

responsible for the induction of cell syncytium formation

and apoptosis, is rapidly degraded in host cells. However,

the mechanism of p10 degradation and its relevance are

still unclear. We report here the identification of cellular

lysosome-associated membrane protein 1 (LAMP-1) as an

interaction partner of p10 by yeast two-hybrid screening,

immunoprecipitation and confocal microscopy assays. We

found that rapid degradation of p10 was associated with

ubiquitination. Importantly, ARV p10 degradation in host

cells could be completely abolished by knockdown of

LAMP-1 by siRNA, indicating that LAMP-1 is required for

ARV p10 degradation in host cells. In contrast, overex-

pression of LAMP-1 facilitated p10 degradation. Further-

more, knockdown of LAMP-1 allowed p10 accumulation,

enhancing p10-induced apoptosis and viral release. Thus,

LAMP-1 plays a critical role in ARV p10 degradation

associated with inhibition of apoptosis and viral release.

Abbreviations

ARV Avian reovirus

LAMP-1 Lysosome-associated membrane protein 1

RNAi RNA interference

Introduction

Avian reovirus (ARV) causes viral arthritis, chronic respi-

ratory diseases, retarded growth, and malabsorption syn-

drome, leading to considerable losses to the poultry

industry. ARV, a member of the genus Orthoreovirus,

family Reoviridae [4], is an icosahedral nonenveloped virus

with a double-protein capsid shell and a genome consisting

of 10 double-stranded RNA segments (L1, L2, L3, M1, M2,

M3, S1, S2, S3, and S4) that express at least 12 primary

translation products, including structural and nonstructural

proteins [4, 24, 40]. The viral proteins encoded by the

L-class genes are designated lambda (k); those encoded by

the M-class, mu (l); and those encoded by the S-class,

sigma (r) [4]. The structural proteins of ARV within each

class have been assigned alphabetical subscripts, such as

kA, kB and kC, to distinguish them from their mammalian

reovirus counterparts, which have been assigned numerical

subscripts (k1, k2, etc.) [4]. There are at least 10 different

structural proteins in the avian reovirus virion, eight of

which (kA, kB, kC, lA, lB, rA, rB and rC) are primary

translation products of their encoded mRNAs, whereas the

other two, lBN and lBC, originate by post-translational

cleavage of their precursor lB [4, 40]. In addition to the

structural proteins, ARV expresses several nonstructural

proteins, such as lNS and rNS, which are encoded by the

M3 and S4 gene, respectively [4, 33, 39], and p10 and p17,

which are encoded by the first two cistrons of the tricistronic

S1 gene [5, 35].

& Yongqiang Wang

vetwyq@cau.edu.cn

& Shijun J. Zheng

sjzheng@cau.edu.cn

1 State Key Laboratory of Agrobiotechnology, China

Agricultural University, Beijing 100193, China

2 Key Laboratory of Animal Epidemiology and Zoonosis,

Ministry of Agriculture, China Agricultural University,

Beijing 100193, China

3 College of Veterinary Medicine, China Agricultural

University, 2 Yuan-Ming-Yuan West Road, Beijing 100193,

China

123

Arch Virol (2016) 161:899–911

DOI 10.1007/s00705-015-2731-5

Reovirus infection induces apoptosis [11, 12, 34], cell-

cell fusion, and syncytium formation [1, 32, 37, 38]. The

ability of ARV to induce apoptosis is not restricted to a

particular virus strain or to a specific cell type, since dif-

ferent ARV isolates were able to induce apoptosis in sev-

eral avian and mammalian cell lines [24]. Although it was

found that reovirus-induced apoptosis was associated with

activation of NF-kappa B [14, 27] and P53 [9, 26, 29],

Sigma C of ARV S1133 and the p10.8 protein of Muscovy

duck reovirus (MDRV) were found to induce apoptosis in

cultured cells [17, 34, 41]. Our recent data show that cel-

lular eukaryotic elongation factor 1 alpha 1 (eEF1A1)

interacts with rC and plays critical roles in rC-inducedapoptosis and inhibition of viral growth [41]. However, the

exact mechanism underlying ARV-induced apoptosis is not

fully understood.

The reovirus fusion-associated small transmembrane

(FAST) proteins, including p10 of ARV and Nelson Bay

reovirus, p14 of reptilian reovirus, are virus-encoded

membrane fusion proteins that function as cell-cell fuso-

gens [1, 15, 38]. ARV p10 induces extensive syncytium

formation in transfected cells [37]. It was found that ARV

p10-induced syncytium formation triggers an apoptotic

response that contributes to altered membrane integrity

[32], suggesting that p10 is not only a cell-cell fusogen but

also an apoptosis-inducer, which may relate to viral repli-

cation and release. P10-induced cell-cell fusion is restricted

by rapid degradation of the majority of newly synthesized

p10 [37]. It is likely that degradation of ARV p10 might be

one of the essential mechanisms employed by host cells to

control ARV infection.

Although ARV p10, a membrane fusion protein and an

apoptosis inducer [32], is subjected to rapid and extensive

degradation [37], the mechanism of p10 degradation in host

cells and the relevance of this biological process are still

unclear. In this study, we found that the cellular protein

LAMP-1 interacts with p10 and is involved in its degrada-

tion. We found that the degradation of p10 could be

inhibited by the inhibitor MG132 and is associated with

ubiquitination. Importantly, ARV p10 degradation in host

cells could be completely abolished by knockdown of

LAMP-1 by siRNA. In contrast, p10 degradation was

enhanced by overexpression of LAMP-1. These data clearly

demonstrate that LAMP-1 plays a critical role in ARV p10

degradation associated with inhibition of viral release.

Materials and methods

Cells and virus

DF-1 (an immortalized chicken embryo fibroblast cell

line), BHK-21 and HEK293T cells were all obtained from

ATCC. All cells were cultured in Dulbecco’s modified

Eagle’s medium (DMEM) (Invitrogen, USA) supple-

mented with 10 % fetal bovine serum (FBS) in a 5 % CO2

incubator. ARV S1133 was kindly provided by Dr. Jin-

gliang Su (China Agricultural University, Beijing, China).

Reagents

All restriction enzymes were purchased from NEB (USA).

pRK5-flag, pEGFP-C1 and pDsRed-monomer-N1 vectors

were obtained from Clontech (USA). DMEM, OPTI-MEM,

RNAiMAX and Lipofectamine LTX were purchased from

Invitrogen (USA). Transfection reagent Vigofect was pur-

chased from Vigorous (Beijing, China). 40,6-diamino-2-

phenylindole (DAPI) and Lyso-tracker Red (C1046) were

purchased from Beytime Company (Nanjing, China). Anti-

FLAG (F1804) antibody was purchased from Sigma (USA),

and anti–green fluorescent protein (anti-GFP; sc-9996), anti-

b-actin (sc-1616-R), anti-HA (Y-11) antibodies were pur-

chased from Santa Cruz Biotechnology (USA). Rabbit anti-

LAMP-1 polyclonal antibody (ab24170)was purchased from

Abcam (UK). FITC-conjugated goat anti-rabbit IgG,TRITC-

conjugated goat anti-mouse IgG, HRP-conjugated goat anti-

mouse and anti-rabbit antibodies were purchased from

DingGuo (Beijing, China). Anti-ARV p10 (EU-0210) and

anti-ARV rC (EU-0211) monoclonal antibodies were pur-

chased from CAEU Biological Company (Beijing, China).

Constructs

ARV p10 was cloned from ARV S1133 using specific

primers (sense primer, 50-atgctgcgtatgcctcccgg-30; anti-

sense primer, 50-tcaaacgtcgtatggcggag-30) (GenBank ID:

AF330703). The chicken lamp-1 gene was originally

cloned from DF-1 cells using specific primers (sense pri-

mer, 50-atgggcggcgctgctcgggccgtcc-30; antisense primer,

50-ttaaatggtttggtacccagcgtgg-30) (GenBank ID:

NM_205283). pRK5-flag-p10, pDsRed-p10, pEGFP-C1-

p10 and pEGFP-C1-lamp-1 were constructed by standard

molecular biology techniques. All the primers were syn-

thesized by Sangon Company (Shanghai, China).

Cell culture and transfection

BHK-21, HEK293T and DF-1 cells were seeded in 6- or

24-well plates. Twenty-four hours later, cells were trans-

fected with plasmids using transfection reagents Vigofect

or Lipofectamine LTX.

Apoptosis assay

DF-1 cells (6 9 105) were seeded on six-well plates and

cultured overnight. Cells were transfected with pEGFP-C1,

900 H. Wu et al.

123

pEGFP-p10 or pEGFP-sigma C. Twenty-four hours after

transfection, cells were trypsinized and stained with

?propidium iodide? (PI) (1 lg/ml) for 10 min at room

temperature. Suspended cells were then analyzed by flow

cytometry. GFP-positive cells were gated for apoptosis

analysis. FACS data were analyzed using CellQuest (BD,

USA).

Yeast two-hybrid screen and colony-life filter assay

A yeast two-hybrid screen was performed according to the

manufacturer’s protocol (Matchmaker Two-Hybrid System

3). The pGBKT7-p10 plasmid expressing the fusion pro-

tein GAL4-binding domain-p10 was used as bait and

introduced by transformation into Saccharomyces cere-

visiae AH109, which is Trp- and contains HIS3, ADE2 and

LacZ reporter genes for GAL4 transcriptional activity.

Chicken cDNA expression library fusion to the GAL4-

activation domain in the pGADT7 plasmid was introduced

by transformation into the Saccharomyces cerevisiae

Y187, which is Leu- and contains the lacZ reporter genes.

In the b-Gal colony-lift filter assay, the bait plasmid was

shown to have no transactivation activity. The cDNA

library clones expressing the interacting prey proteins were

screened by yeast mating. Positive clones were selected on

SD/-Ade/-His/-Leu/-Trp medium and tested for b-galac-tosidase activity (LacZ?) by colony-lift filter assay. Yeast

transfected with pGBKT7-p53 and pGADT7-T was used as

a positive control, and yeast transfected with pGBKT7-

Lam and pGADT7-T was used as a negative control for the

blue color reaction. The resulting clones were sequenced

with the GAL4-AD sequencing sense primer 5’-

AGATGGTGCACGATGCACAG-3’, and the results were

subjected to a BLAST search against the NCBI database.

Immunoprecipitation and western blot

For immunoprecipitation, BHK-21 or DF-1 cells (6 9105)

were seeded in 6-well plates and cultured for 24 h before

co-transfection with pRK5-flag-p10 or pRK5-flag and

pEGFP-C1 or pEGFP-lamp-1. Twenty-four hours after

transfection, cell lysates were prepared using a lysis buffer

(1 % Triton X-100, 20 mM Tris-Cl, 5 mM EDTA,

137 mM NaCl, 0.02 % NaN3, and 1 % protease inhibitor

cocktail C). The cell lysates were incubated with 2 lg of

anti-FLAG antibody at 4 �C for 2 h and then mixed with

20 lL of a 50 % slurry of protein A/G PLUS-Agarose and

incubated for another 2 h. Beads were washed three times

with the lysis buffer and boiled with 29 SDS loading

buffer for 10 min. The samples were fractionated by

electrophoresis on 10 % SDS-PAGE gels, and the resolved

proteins were transferred to PVDF membranes. After

blocking with 5 % skimmed milk, the membranes were

incubated with either anti-GFP or anti-FLAG antibodies,

followed by the appropriate HRP-conjugated secondary

antibody. Blots were developed using an enhanced

chemiluminescence (ECL) kit. For endogenous LAMP-1

pull-down assay, DF-1 cells were transfected with pRK5-

flag-p10 or empty vector. Twelve hours after transfection,

the cell lysates were subjected to immunoprecipitation with

anti-FLAG antibody and immunoblotted with anti-LAMP-

1 or anti-FLAG antibodies. For the endogenous LAMP-1

pull-down assay in ARV-infected cells, DF-1 cells were

infected with ARV at an MOI of 10. Twenty-four hours

postinfection, cell lysates were prepared and immunopre-

cipitated with anti-p10 antibody and immunoblotted with

anti-p10, anti-LAMP-1 or anti-rC antibodies.

Confocal laser scanning microscopy assay

DF-1 cells (1 9 105) were seeded on coverslips in 24-well

plates and were cultured overnight before transfection with

pEGFP-lamp-1 and pDsRed-p10. Twenty-four hours after

transfection, cells were fixed with 4 % paraformaldehyde,

and the nuclei were stained with DAPI. The cell samples

were observed using a laser confocal scanning microscope.

Immunofluorescence antibody assay (IFA)

DF-1 cells were mock infected or infected with ARV at an

MOI of 10. Twenty-four hours after infection, cells were

fixed with 4 % paraformaldehyde, permeabilized with

0.2 % Triton X-100, blocked with 1 % bovine serum

albumin, and incubated with anti-p10 monoclonal antibody

and rabbit anti-LAMP-1 antibodies, followed by TRITC-

conjugated goat anti-mouse antibodies (red) and FITC-

conjugated goat anti-rabbit antibodies (green). Nuclei were

counterstained with DAPI (blue). The cell samples were

observed using a laser confocal scanning microscope.

P10 half-life and ubiquitination assays

DF-1 cells (6 9 105) were seeded in 6-well plates. Twenty-

four hours later, cells were transfected with pRK5-flag-p10.

Twelve hours after transfection, cells were treated with

50 lg of cycloheximide (CHX) per ml for the indicated

time. The cell lysates were prepared at different time points

and analyzed for the expression of proteins by Western

blot. The level of p10 was quantified by densitometry and

normalized to that of b-actin. For the ubiquitination assay,

HEK293T cells were transfected with pRK5-flag-p10 or

pRK5-flag and pCMV-HA-Ub. Cells were treated with

MG132 for 4 h and lysed in 1 % SDS. After boiling for

10 min, lysates were diluted 10 times with cold lysis buffer

supplemented with 19 complete inhibitor and 10 mM

N-ethylmaleimide (NEM). Cell samples were subjected to

Role of LAMP-1 in avian reovirus P10 degradation 901

123

immunoprecipitation with anti-FLAG antibody and then

analyzed by Western blot. Ubiquitinated p10 was probed

by anti-HA antibody.

Knockdown of LAMP-1 by RNAi

The siRNA was designed by Genechem Company

(Shanghai, China) and used to knockdown LAMP-1 in DF-

1 cells. The sequences of siRNA for targeting LAMP-1 in

DF-1 cells were as follows: RNAi#1 (sense, 50-GCAGUUUGCACACUUCUUU-30; antisense, 50-AAAGAAGUGUGCAAACUGC-30), RNAi#2 (sense, 50-GCUCGUGUGGUGAAGGUAA-30; antisense, 50-UUACCUUCACCUCUCGAGC-30), RNAi#3 (sense, 50-GGUAACACAUCUCAUCCAA-30; antisense, 50-UUGGAUGAGAUGUGUUACC-30), negative control (sense: 5’-UUCUCCGAACGUG

UCACGUtt-3’; antisense: 5’-AUCUCUAUCCAGCAUU

GAtt-3’). DF-1 cells (2 9 105) were seeded in 12-well

plates and cultured for 24 h before transfection with siRNA

using RNAi MAX as per the manufacturer’s instructions

(Invitrogen, USA). Double transfections were performed at

a 24-h interval. Twenty-four hours after the second trans-

fection, cells were harvested for further analysis.

Examination of syncytium formation

DF-1 cells were mock infected or infected with ARV

S1133 at an MOI of 10. Twenty-four hours after ARV

infection, cells were fixed with 4 % paraformaldehyde and

stained with Giemsa stain to visualize syncytia. Syncytium

formation was examined by microscopy. The syncytial

nuclei in each microscopic field were counted, and the

average number of syncytial nuclei from three fields was

calculated.

Measurement of ARV replication in DF-1 cells

Untreated cells or cells receiving LAMP-1-specific siR-

NAs or control siRNA were infected with ARV at an

MOI of 10. Cell cultures were collected at different time

points (12, 24, 36, and 48 h) after infection. The culture

samples were freeze-thawed three times and centrifuged

at 2000 9 g for 10 min. The viral contents in the super-

natants and cell culture were titrated using TCID50 (50 %

tissue culture infective doses) in DF-1 cells. Briefly, the

virus suspension was diluted tenfold in DMEM. A 100-lLaliquot of each diluted sample was added to each well of

a 96-well plate, followed by addition of 100 lL of DF-1

cells at a density of 5 9 105 cells/mL. Cells were cul-

tured for 5 days at 37 �C in 5 % CO2. A cell culture

showing a cytopathic effect (CPE) was considered posi-

tive. The titer was calculated based on a previously

described method [30].

Statistical analysis

The significance of the differences between pEGFP-p10/

rC-transfected cells and controls in induction of apoptosis

and between LAMP-1-RNAi cells and controls in viral

growth was determined by the Mann-Whitney test or

ANOVA, as appropriate.

Results

Transfection of DF-1 cells with pEGFP-p10 induces

apoptosis

Since ARV p10 is membrane fusion protein that is

responsible for syncytium formation, which triggers

apoptosis [32] and ARV sigmaC induces apoptosis in

BHK-21 and Vero cells [34], we examined the apoptotic

effect of p10 by transient expression of a p10-GFP fusion

construct in DF-1 cells using rC as a parallel control. We

transfected DF-1 cells with pEGFP-p10 or pEGFP-rC and

examined apoptotic cells by flow cytometry using PI

staining. As shown in Fig. 1A and B, GFP was evenly

distributed in the cytoplasm of pEGFP-C1-transfected

cells. However, when cells were transfected with pEGFP-

p10 or pEGFP-rC alone, GFP-p10 or GFP-rC fusion

proteins were expressed and made a speckled pattern

(Fig. 1C-F). These fusion proteins induced apoptosis in

DF-1 cells (Fig. 1G) (p\ 0.05). The apoptosis induced by

GFP-p10 was comparable to that of GFP-rC, suggestingthat p10 is not only a membrane fusion protein but also an

apoptosis inducer in cells.

P10 interacts with the cellular protein LAMP-1

After the identification of p10 as the viral protein inducing

cell death in DF-1 cells, we investigated the mechanism of

this induction by searching for its cellular targets. To this

end, we used p10 as bait in the yeast two-hybrid system to

screen a cDNA library generated from the spleen of a

chicken. Among the proteins that potentially interact with

p10, the LAMP-1 protein was identified five times. This

protein might be relevant to p10 function because it is

involved in apoptosis [8, 13, 23]. Thus, we constructed a

plasmid that allows the expression of GFP-LAMP-1 to

analyze its interaction with p10 in BHK-21 and DF-1 cells.

When lysates of cells expressing both Flag-p10 and GFP-

LAMP-1 were immunoprecipitated with FLAG antibody,

GFP-LAMP-1 was detected in the precipitate, indicating

that p10 interacted with ectopically expressed LAMP-1 in

BHK21 cells (Fig. 2A). Similar results were obtained in

experiments using the DF-1 cells (Fig. 2B), demonstrating

that the observed interaction between these two proteins is

902 H. Wu et al.

123

not cell type specific. To confirm that p10 binds to LAMP-

1, we expressed p10 in DF-1 cells and examined its

interaction with endogenous LAMP-1 using a pull-down

assay. The binding of FLAG-p10 with endogenous LAMP-

1 was readily detectable in cells expressing the viral protein

(Fig. 2C). Furthermore, we examined the interaction of

ARV p10 with endogenous LAMP-1 in ARV-infected

cells. Likewise, LAMP-1 was pulled down from the lysates

of ARV-infected cells by anti-p10 antibodies, but not from

mock-infected controls, indicating that ARV p10 interacts

with LAMP-1 in ARV-infected cells (Fig. 2D). These

results clearly show that ARV p10 interacts with LAMP-1

in host cells.

P10 colocalizes with LAMP-1 in host cells

To determine the subcellular localization of p10 and

LAMP-1, we performed confocal microscopy assay with

BHK-21 cells transfected to express DsRed-p10 and GFP-

LAMP-1. Transfection of BHK-21 cells with pEGFP-

LAMP-1 or pDsRed-p10 indicated that both LAMP-1 and

p10 were located in cytoplasm (Fig. 3A and B). When cells

were transfected with both plasmids, we observed colo-

calization of LAMP-1 with p10 in the transfected cells

(Fig. 3C-E). To determine whether endogenous LAMP-1

colocalizes to the same cellular compartment, we

expressed DsRed-p10 in DF-1 cells by transfection.

Transfected samples were immunostained with anti-

LAMP-1 antibody and an FITC-conjugated goat anti-rabbit

antibody. Consistent with the above observation, the

endogenous LAMP-1 was also co-localized with p10 in the

cytoplasm (Fig. 3F-H). To corroborate our findings that

LAMP-1 colocalizes with p10, we examined the interaction

of p10 with LAMP-1 in ARV-infected cells using

immunofluorescence antibody assay (IFA). We infected

DF-1 cells with ARV at an MOI of 10 and performed IFA

using anti-p10 and anti-LAMP-1 antibodies. The results

showed that endogenous LAMP-1 was also co-localized

with p10 in ARV-infected cells (Fig. 3I-N). In addition,

transfection of BHK-21 cells with pEGFP-p10 indicated

that p10 was localized with lysosomes (Fig. 3O-Q).

LAMP-1, as indicated by its name, is a lysosomal-associ-

ated membrane protein, and our data suggest that p10

interacts with LAMP-1 in the lysosome.

P10 is degraded via the ubiquitination-dependent

proteasomal pathway

Because p10 interacts with LAMP-1 (Fig. 2 and 3) and it

also subjected to rapid and extensive degradation [32, 37],

we investigated how p10 is degraded in cells and whether

its degradation is associated with LAMP-1. We transfected

Fig. 1 Transfection of DF-1 cells with pEGFP-p10 induces apopto-

sis. (A-F) Expression of GFP-p10 and -rC fusion proteins in DF-1

cells. DF-1 cells (6 9 105) were seeded in six-well plates and

cultured overnight. Cells were transfected with pEGFP-C1, pEGFP-

p10 and pEGFP-sigma C plasmids. Twenty-four hours after trans-

fection, cells were observed by a fluorescence microscopy. (G) Per-

centage of apoptotic cells after transfection with the indicated

expression plasmids. Flow cytometry analysis of apoptosis in

transfected DF-1 cells expressing GFP, GFP-p10, and -rC fusion

proteins. DF-1 cells were transfected with different plasmids as

described above. Twenty-four hours after transfection, cells were

harvested and stained with PI, followed by analysis with flow

cytometry. GFP-positive cells were gated for further analysis of PI-

staining-positive cells. Results are representative of three independent

experiments. Data are represented as mean ?/- SD, n = 3. **,

p\ 0.01; *, p\ 0.05

Role of LAMP-1 in avian reovirus P10 degradation 903

123

cells with pRK5-flag-p10 and continuously cultured the

cells for 12 hours before treatment with cycloheximide

(CHX), a protein synthesis inhibitor. Consistent with a

previous publication [37], p10 was rapidly degraded after

CHX treatment (Fig. 4A and B), suggesting that p10 has a

short half-life in cells. However, when cells expressing

Flag-p10 were cultured in the presence of both CHX and

the proteasome inhibitor MG132, p10 degradation was

markedly inhibited, indicating that p10 might be degraded

within the proteasome (Fig. 4C and D). To determine

whether p10 is subjected to ubiquitination, we transfected

cells with both pRK5-flag-p10 and HA-linked ubiquitin

(HA-Ub) expression vectors in the presence of MG132. As

a result, the flag-p10 fusion protein was ubiquitinated

(Fig. 4E). Taken together, our results suggest that p10 is

targeted for ubiquitin-proteasomal degradation in cells.

LAMP-1 is required for p10 degradation

The fact that p10 induces apoptosis, is subjected to ubiq-

uitin-proteasomal degradation, and interacts with LAMP-1

suggests that LAMP-1 plays a role in p10-induced apop-

tosis and/or in its degradation and that knockdown of

LAMP-1 would therefore affect apoptosis and/or degra-

dation of p10. To test this hypothesis, we made three

LAMP-1 RNAi constructs and found that one of them

could effectively lower the cellular level of LAMP-1

without causing discernable changes in cell morphology

(Fig. 5A). We then infected DF-1 cells receiving this

siRNA or control siRNA with ARV S1133 at an MOI of 10

and examined the expression of p10 in these cells after

infection with ARV, using b-actin and rC, a structural

protein encoded by the S1 gene of ARV, as internal con-

trols. Surprisingly, degradation of p10 in ARV-infected

cells was completely abolished by knockdown of LAMP-1,

while ARV rC expression was not affected (Fig. 5B). In

contrast, overexpression of LAMP-1 significantly reduced

the amount of p10 in pRK-5-flag-p10-transfected cells

(Fig. 6A-F). These data clearly demonstrate that LAMP-1

is required for p10 degradation in cells.

Fig. 2 The interaction of ARV p10 with the host-cell protein LAMP-

1. (A and B) Interaction of p10 with exogenous LAMP-1. BHK-21

(A) and DF-1 cells (B) were transfected with the indicated expression

plasmids. Sixteen hours after transfection, cell lysates were prepared

and immunoprecipitated (IP) with anti-FLAG antibody and

immunoblotted with anti-FLAG or anti-GFP antibodies. (C) Interac-

tion of p10 with endogenous LAMP-1. DF-1 cells were transfected

with pRK5-FLAG-p10 or empty vector as a control. Twelve hours

after transfection, cell lysates were prepared and immunoprecipitated

with anti-FLAG antibody and immunoblotted with anti-FLAG or anti-

LAMP-1 antibodies. (D) Interaction of ARV p10 with endogenous

LAMP-1 in ARV-infected cells. DF-1 cells were infected with ARV

S1133 at an MOI of 10. Twenty-four hours post infection, cell lysates

were prepared and immunoprecipitated with anti-p10 antibody and

immunoblotted with anti-p10 or anti-LAMP-1 antibodies

cFig. 3 Colocalization of p10 with LAMP-1 in cells. (A-E) Localiza-

tion of p10 and exogenous LAMP-1 in BHK-21 cells. BHK-21 cells

(2 9 105) were seeded in 24-well plates with coverslips in the wells

and cultured overnight. Cells were transfected with pEGFP-LAMP-1

(A) or pDsRed-p10 (B) or transfected with both pDsRed-p10 and

pEGFP-LAMP-1 (C-E). Sixteen hours after transfection, cells were

fixed with 4 % paraformaldehyde. After washing, the cell nuclei were

counterstained with DAPI (blue). The cell samples were observed

using a laser confocal scanning microscope. (F-H) Colocalization of

p10 with endogenous LAMP-1 in DF-1 cells. DF-1 cells were

transfected with pDsRed-p10. Sixteen hours after transfection, cells

were fixed with 4 % paraformaldehyde. After washing, the fixed cells

were permeabilized with 0.2 % Triton X-100 and immunostained

with anti-LAMP-1 and FITC-conjugated secondary antibodies. Nuclei

were counterstained with DAPI (blue). The cell samples were

observed using a laser confocal scanning microscope. (I-N) Colocal-

ization of ARV p10 with endogenous LAMP-1 in ARV-infected cells.

DF-1 cells were mock infected or infected with ARV at an MOI of 10.

Twenty-four hours after infection, cells were fixed and probed with

mouse anti-p10 and rabbit anti-LAMP-1 antibodies, followed by

TRITC-conjugated goat anti-mouse antibody (red) and FITC-conju-

gated goat anti-rabbit antibody (green). Nuclei were counterstained

with DAPI (blue). The cell samples were observed using a laser

confocal scanning microscope. (O-Q) localization of p10 in the

lysosome. BHK-21 cells were transfected with pEGFP-p10 plasmid.

Twenty-four hours after transfection, cells were stained with

LysoTracker Red for the lysosome and observed under a laser

confocal scanning microscope (color figure online)

904 H. Wu et al.

123

Role of LAMP-1 in avian reovirus P10 degradation 905

123

Knockdown of LAMP-1 enhances p10-induced

apoptosis and ARV-induced syncytium formation

in cells

Since LAMP-1 is required for p10 degradation in cells and

p10 induces apoptosis and mediates cell-cell membrane

fusion [2, 10, 32], we proposed that accumulation of p10 in

cells by LAMP-1 knockdown would therefore enhance

p10-induced apoptosis and ARV-induced syncytium for-

mation. To test this hypothesis, we knocked down LAMP-1

in DF-1 cells and transfected these cells with pEGFP-p10

or infected them with ARV 1133, followed by flow-cy-

tometry analysis of apoptotic cells using annexin-V and

7-AAD staining or histochemical examination of syn-

cytium formation using Giemsa staining. We observed that

the GFP-p10 fusion protein accumulated in LAMP-1

knockdown cells, but the levels of this protein were much

reduced in controls (Fig. 7A). Importantly, knockdown of

LAMP-1 markedly enhanced p10-induced apoptosis

(Fig. 7B) and ARV-induced syncytium formation (7C-I)

when compared to RNAi controls (p\ 0.05). These results

strongly suggest that knockdown of LAMP-1 allows p10

Fig. 4 P10 is degraded by ubiquitin-dependent proteasome. (A and

B) p10 is rapidly degraded in cells. (A) DF-1 cells (6 9 105) were

seeded in six-well plates and cultured overnight. Cells were

transfected with pRK5-FLAG-p10 plasmid. Twelve hours after

transfection, cells were incubated with 50 lg of cycloheximide

(CHX) per ml for the indicated time. The cell lysates were prepared at

different time points and the expression level of proteins was

analyzed by Western blot. (B) The level of p10 in (A) was quantified

by densitometry and normalized to that of actin. The value of

normalized p10 at time 0 h was set at 1.0. (C) Effect of MG132 on

p10 degradation. DF-1 cells (6 9 105) were seeded in six-well plates

and cultured overnight. Cells were transfected with pRK5-FLAG-p10.

Twelve hours after transfection, cells were treated with or without

20 lMMG132, and with 50 lg of CHX per ml for the indicated time.

Cell lysates were prepared at different time points, and the level of

proteins was analyzed by Western blot. (D) The levels of p10 in

(C) were quantified by densitometry and normalized to that of actin.

The value of normalized p10 at time 0 h was set at 1.0. (E) P10 is

ubiquitinated in cells. HEK293T cells were cotransfected with FLAG-

tagged p10 and HA-linked ubiquitin (HA-Ub) expression vector.

Twenty-four hours after transfection, cells were treated with 20 lMMG132 for 4 h. Cell lysates were immunoprecipitated with anti-

FLAG antibody and immunoblotted with anti-HA and anti-FLAG

antibodies

906 H. Wu et al.

123

accumulation, which enhances p10-induced apoptosis and

ARV-induced syncytium formation.

Knockdown of LAMP-1 facilitates ARV release

Since LAMP-1 is required for ARV p10 degradation, we

hypothesized that LAMP-1 might play a critical role in the

anti-ARV response in host cells and that knockdown of

LAMP-1 would therefore facilitate ARV growth. To test

this hypothesis, we examined viral replication in LAMP-1

knockdown cells by measuring viral loads in ARV S1133-

infected DF-1 cell cultures at different time points postin-

fection. Consistent with its postulated role in the antiviral

response, cell culture supernatants with lower LAMP-1

levels had higher ARV loads than the RNAi control

(p\ 0.05) (Fig. 8A), but the viral loads in cell cultures

were similar between LAMP-1 RNAi and RNAi control

(Fig. 8B). These results indicate that LAMP-1 might be

involved in the anti-ARV response in cells via facilitating

ubiquitin-proteasomal degradation of p10, which triggers

apoptosis and ARV release.

Discussion

ARV causes viral arthritis, chronic respiratory diseases,

retarded growth and malabsorption syndrome, leading to

considerable losses to the poultry industry. ARV infection

induces apoptosis in cultured cells [11, 24, 31, 41]. One of

the reovirus proteins, the ARV rC protein, was found to

induce apoptosis in cells [34]. In a recent publication,

sigma1s (r1S), a nonstructural protein encoded by S1

gene, was also identified as an apoptosis inducer [6]. In a

previous study, we found that ARV rC induced apoptosis

via interaction with cellular eEF1A1, which restricts viral

release [41]. The present study shows that expression of

p10 induces apoptosis in DF-1 cells to the same extent as

that of rC, suggesting that p10 may contribute to ARV-

induced apoptosis. This observation supports the previous

observation that ARV-p10-induced syncytium formation

triggers an apoptotic response contributing to altered

membrane integrity [32]. As p10-induced cell-cell fusion is

restricted by rapid degradation of the majority of newly

synthesized p10 [37], it is likely that degradation of ARV

P10 might be one of the essential mechanisms employed by

host cells to control ARV growth.

A series of studies have been carried out to elucidate

how p10 induces cell-cell fusion, and much progress has

been made [2, 10, 22, 36, 38]. However, little information

is available about how p10 is degraded in cells. In the

present study, we found that the rapid degradation of p10

could be inhibited by MG132 and that the proteasomal

degradation of p10 involved ubiquitination. Importantly,

the rapid degradation of p10 could be inhibited by

knockdown of LAMP-1. In contrast, overexpression of

LAMP-1 significantly reduced p10 levels in cells. Clearly,

LAMP-1 is required for the proteasomal degradation of

ARV p10. One of the features that make proteins short-

lived is surface hydrophobicity [7], and p10 is such a

molecule, containing a hydrophobic patch (HP) [2]. It has

been reported that the HP allows specific recognition of the

polyubiquitin degradation signal by the 26S proteasome

[3]. Thus, it is very likely that p10 HP is necessary for

ubiquitin-mediated proteasomal degradation. Our data

show that p10 was markedly ubiquitinated, indicating

ubiquitin-mediated proteasomal degradation of p10 in

cells.

These observations raise several questions. For example,

what kind of signaling events are triggered by LAMP-1

Fig. 5 Knockdown of LAMP-1 results in accumulation of p10 in

cells. (A) Effect of LAMP-1 RNAi on the expression of endogenous

LAMP-1. DF-1 cells were transfected with siRNA (RNAi#1-3) or

controls. Forty-eight hours after the second transfection, cell lysates

were prepared and examined by Western blot with anti-LAMP-1

antibody. Endogenous b-actin expression was used as an internal

control. (B) Knockdown of LAMP-1 resulted in accumulation of

ARV p10. Normal DF-1 cells, control RNAi cells, and LAMP-1

RNAi cells were infected with ARV at an MOI of 10. Twenty-four

hours postinfection, cell lysates were prepared, and the expression of

ARV p10 and rC was examined by Western blot using anti-p10 and

anti-rC monoclonal antibodies

Role of LAMP-1 in avian reovirus P10 degradation 907

123

upon interaction with p10? Does LAMP-1 directly initiate

p10 degradation in the proteasome? What role does the

lysosome play in p10 degradation, since LAMP-1 is a

membrane protein of the lysosome? More efforts will be

required to investigate how LAMP-1 is involved in p10

degradation.

LAMP-1 plays a critical role in the proteasomal degra-

dation of ARV p10, which might be one of the defense

mechanisms employed by host cells to control ARV

infection. Our data show that knockdown of LAMP-1

allows p10 accumulation, enhancing p10-induced apopto-

sis, ARV-induced syncytium formation, and viral release.

This is in agreement with the proposal that p10, a fusion-

associated small transmembrane (FAST) protein of the

fusogenic reoviruses, promotes localized cell-cell trans-

mission of the infection followed by enhanced progeny

virus release from apoptotic syncytia and systemic dis-

semination of the infection [32].

LAMP-1 is one of the major lysosomal membrane

proteins. LAMP-1 and LAMP-2, also called CD107a and

CD107b, respectively, constitute about 50 % of all proteins

of the lysosome membrane [13, 16, 23]. LAMP-1 and

LAMP-2 share some common functions in vivo [16], but

each of them may have specific functions [13, 23]. It has

been reported that LAMP-1 was involved in perforin-in-

duced apoptosis of NK cells, because knockdown of

LAMP-1 in primary human natural killer (NK) cells and

deficiency of LAMP-1 in mice resulted in increased NK

cell apoptosis upon target-cell-induced degranulation [13,

23]. The evidence indicates that LAMP-1 might be

involved in apoptosis [8]. Our data show that knockdown

of LAMP-1 abolished proteasomal degradation of ARV

p10, leading to accumulation of p10 in cells, which may

directly contribute to p10-induced apoptosis and ARV-in-

duced syncytium formation associated with facilitation of

viral release. Thus, it is likely that LAMP-1 acts as an anti-

Fig. 6 Overexpression of LAMP-1 reduces the protein level of p10 in

cells. (A-D) Effect of LAMP-1 on p10 expression in pRK5-FLAG-

p10-transfected DF-1 cells. DF-1 cells (6 9 105) were seeded in six-

well plates and cultured overnight. Then, cells were transfected with

both pRK5-FLAG-p10 and pEGFP-C1 empty vector (A and B) or

pEGFP-LAMP-1 (C and D). Sixteen hours after transfection, the cell

samples were observed under a fluorescence microscope. (E and F)

Effect of LAMP-1 on p10 as examined by Western blot. DF-1 cells

(6 9 105) were seeded in six-well plates and cultured overnight. Then

cells were transfected with expression plasmids as described above

(A-D). Sixteen hours after transfection, cell lysates were prepared and

examined by Western blot using anti-Flag and anti-GFP antibodies

(E). The level of p10 expression was quantified by densitometry and

normalized to that of b-actin (F)

908 H. Wu et al.

123

defense molecule in the host in the sense that it mediates

proteasomal degradation of p10 to reduce apoptosis and

viral release.

Of note, comparative sequence analysis has shown that

there is extensive sequence divergence between ARV and

other orthoreoviruses [21]. It has been found that ARV

demonstrates a significant polymorphism in the migration

pattern of the dsRNA segments among different isolates

[18, 19], and the diversity within the P1-encoding gene is

neither dependent on the viral serotype nor correlated with

the disease state caused by ARV [21]. Our data were

obtained from experiments using ARV S1133, a strain that

is commonly used for studies of ARV [5, 20, 24, 25, 28, 39,

40]. More efforts will be required to investigate the

diversity of functional domains of p10 among ARV strains.

In summary, our data show that p10 induces apoptosis

and specifically interacts with cellular LAMP-1. P10 is

degraded by the ubiquitin-proteasomal machinery, and the

degradation of p10 can be inhibited by knockdown of

LAMP-1. Furthermore, overexpression of LAMP-1 sig-

nificantly reduces p10 levels in cells. Moreover, knock-

down of LAMP-1 allows p10 accumulation in cells,

Fig. 7 Knockdown of LAMP-1 allows p10 accumulation associated

with enhancement of p10-induced apoptosis and ARV-induced

syncytium formation in cells. (A) Knockdown of LAMP-1 by RNAi

allows accumulation of GFP-p10 in cells. DF-1 cells were treated

with LAMP-1 RNAi or control RNAi and then transfected with

pEGFP-p10 or pEGFP-C1 empty vector. Twenty-four hours after

transfection, cell lysates were prepared and subjected to Western blot

analysis. (B) Knockdown of LAMP-1 enhances p10-induced apop-

tosis in cells. Cells were treated as in panel A and stained with

annexin-V-PE and 7-AAD, followed by flow cytometry analysis. (C-

I) Knockdown of LAMP-1 enhances ARV-induced syncytium

formation. DF-1 cells were treated as in panel A and were mock

infected or infected with ARV S1133 at an MOI of 10. Twenty-four

hours postinfection, cells were fixed and stained with Giemsa to

visualize syncytia. The cell samples were observed under a regular

microscope (2009). All scale bars represent 10 lm. (I) The average

number of syncytial nuclei per field was measured. The significance

of the difference between LAMP-1 RNAi and control RNAi

treatments in apoptosis and syncytium formation was analyzed by

ANOVA. Data are the mean ?/- standard deviation of three

independent experiments. *, p\ 0.05; ***, p\ 0.001

Role of LAMP-1 in avian reovirus P10 degradation 909

123

enhancing p10-induced apoptosis and viral release. Thus,

LAMP-1 plays a critical role in ARV p10 degradation

associated with inhibition of apoptosis and viral spread.

Acknowledgments We thank Dr. Jingliang Su for his kind assis-

tance. This work was supported by grants from the National Natural

Science Foundation of China (#31272543 and #31430085) and Ear-

marked Fund for Modern Agro-Industry Technology Research Sys-

tem (#NYCYTX-41).

References

1. Barry C, Duncan R (2009) Multifaceted sequence-dependent and

-independent roles for reovirus FAST protein cytoplasmic tails in

fusion pore formation and syncytiogenesis. J Virol

83:12185–12195

2. Barry C, Key T, Haddad R, Duncan R (2010) Features of a

spatially constrained cystine loop in the p10 FAST protein

ectodomain define a new class of viral fusion peptides. J Biol

Chem 285:16424–16433

3. Beal RE, Toscano-Cantaffa D, Young P, Rechsteiner M, Pickart

CM (1998) The hydrophobic effect contributes to polyubiquitin

chain recognition. Biochemistry 37:2925–2934

4. Benavente J, Martinez-Costas J (2007) Avian reovirus: structure

and biology. Virus Res 123:105–119

5. Bodelon G, Labrada L, Martinez-Costas J, Benavente J (2001)

The avian reovirus genome segment S1 is a functionally tri-

cistronic gene that expresses one structural and two nonstructural

proteins in infected cells. Virology 290:181–191

6. Boehme KW, Hammer K, Tollefson WC, Konopka-Anstadt JL,

Kobayashi T, Dermody TS (2013) Nonstructural protein sigma1s

mediates reovirus-induced cell cycle arrest and apoptosis. J Virol

87:12967–12979

7. Bohley P (1996) Surface hydrophobicity and intracellular

degradation of proteins. Biol Chem 377:425–435

8. Chen JW, Madamanchi N, Madamanchi NR, Trier TT, Keherly

MJ (2001) Lamp-1 is upregulated in human glioblastoma cell

lines induced to undergo apoptosis. J Biomed Sci 8:365–374

9. Chulu JL, Lee LH, Lee YC, Liao SH, Lin FL, Shih WL, Liu HJ

(2007) Apoptosis induction by avian reovirus through p53 and

mitochondria-mediated pathway. Biochem Biophys Res Commun

356:529–535

10. Clancy EK, Duncan R (2009) Reovirus FAST protein trans-

membrane domains function in a modular, primary sequence-

independent manner to mediate cell–cell membrane fusion.

J Virol 83:2941–2950

11. Clarke P, Tyler KL (2003) Reovirus-induced apoptosis: a

minireview. Apoptosis 8:141–150

12. Coffey CM, Sheh A, Kim IS, Chandran K, Nibert ML, Parker JS

(2006) Reovirus outer capsid protein micro1 induces apoptosis

and associates with lipid droplets, endoplasmic reticulum, and

mitochondria. J Virol 80:8422–8438

13. Cohnen A, Chiang SC, Stojanovic A, Schmidt H, Claus M, Saftig

P, Janssen O, Cerwenka A, Bryceson YT, Watzl C (2013) Surface

CD107a/LAMP-1 protects natural killer cells from degranulation-

associated damage. Blood 122:1411–1418

14. Connolly JL, Rodgers SE, Clarke P, Ballard DW, Kerr LD, Tyler

KL, Dermody TS (2000) Reovirus-induced apoptosis requires

activation of transcription factor NF-kappaB. J Virol

74:2981–2989

15. Corcoran JA, Duncan R (2004) Reptilian reovirus utilizes a small

type III protein with an external myristylated amino terminus to

mediate cell–cell fusion. J Virol 78:4342–4351

16. Eskelinen EL (2006) Roles of LAMP-1 and LAMP-2 in lysosome

biogenesis and autophagy. Mol Aspects Med 27:495–502

17. Geng H, Zhang Y, Liu-Partanen Y, Vanhanseng Guo D, Wang Y,

Liu M, Tong G (2009) Apoptosis induced by duck reovirus p10.8

protein in primary duck embryonated fibroblast and Vero E6

cells. Avian Dis 53:434–440

18. Gouvea VS, Schnitzer TJ (1982) Polymorphism of the migration

of double-stranded RNA genome segments of avian reoviruses.

J Virol 43:465–471

19. Gouvea VS, Schnitzer TJ (1982) Polymorphism of the genomic

RNAs among the avian reoviruses. J Gen Virol 61(Pt 1):87–91

20. Hsu CJ, Wang CY, Lee LH, Shih WL, Chang CI, Cheng HL,

Chulu JL, Ji WT, Liu HJ (2006) Development and characteri-

zation of monoclonal antibodies against avian reovirus sigma C

protein and their application in detection of avian reovirus iso-

lates. Avian Pathol 35:320–326

Fig. 8 Knockdown of LAMP-1 facilitates ARV release. (A and B)

DF-1 cells were treated with LAMP-1-RNAi or control-RNAi

constructs or medium only as controls and were infected with ARV

S1133 at an MOI of 10. At different time points (12, 24, 36 and 48 h)

after ARV infection, the viral titers in the supernatants (A) or cell

cultures (B) were determined by TCID50 analysis using 96-well

plates. The significance of the difference between LAMP-1 RNAi and

control RNAi treatments was analyzed by ANOVA. The graph shows

the average of viral titers in DF-1 cells from three independent

experiments

910 H. Wu et al.

123

21. Hsu HW, Su HY, Huang PH, Lee BL, Liu HJ (2005) Sequence

and phylogenetic analysis of P10- and P17-encoding genes of

avian reovirus. Avian Dis 49:36–42

22. Key T, Duncan R (2014) A compact, multifunctional fusion

module directs cholesterol-dependent homomultimerization and

syncytiogenic efficiency of reovirus p10 FAST proteins. PLoS

Pathog 10:e1004023

23. Krzewski K, Gil-Krzewska A, Nguyen V, Peruzzi G, Coligan JE

(2013) LAMP1/CD107a is required for efficient perforin delivery

to lytic granules and NK-cell cytotoxicity. Blood 121:4672–4683

24. Labrada L, Bodelon G, Vinuela J, Benavente J (2002) Avian

reoviruses cause apoptosis in cultured cells: viral uncoating, but

not viral gene expression, is required for apoptosis induction.

J Virol 76:7932–7941

25. Lin HY, Chuang ST, Chen YT, Shih WL, Chang CD, Liu HJ

(2007) Avian reovirus-induced apoptosis related to tissue injury.

Avian Pathol 36:155–159

26. Lin PY, Lee JW, Liao MH, Hsu HY, Chiu SJ, Liu HJ, Shih WL

(2009) Modulation of p53 by mitogen-activated protein kinase

pathways and protein kinase C delta during avian reovirus S1133-

induced apoptosis. Virology 385:323–334

27. Lin PY, Liu HJ, Liao MH, Chang CD, Chang CI, Cheng HL, Lee

JW, Shih WL (2010) Activation of PI 3-kinase/Akt/NF-kappaB

and Stat3 signaling by avian reovirus S1133 in the early stages of

infection results in an inflammatory response and delayed apop-

tosis. Virology 400:104–114

28. Lin PY, Liu HJ, Chang CD, Chang CI, Hsu JL, Liao MH, Lee

JW, Shih WL (2011) Avian reovirus S1133-induced DNA dam-

age signaling and subsequent apoptosis in cultured cells and in

chickens. Arch Virol 156:1917–1929

29. Liu HJ, Lin PY, Lee JW, Hsu HY, Shih WL (2005) Retardation

of cell growth by avian reovirus p17 through the activation of p53

pathway. Biochem Biophys Res Commun 336:709–715

30. Reed LJ, Muench H (1938) A simple method of estimating fifty

per cent endpoints. Am J Epidemiol 27:493–497

31. Rodriguez-Grille J, Busch LK, Martinez-Costas J, Benavente J

(2014) Avian reovirus-triggered apoptosis enhances both virus

spread and the processing of the viral nonstructural muNS pro-

tein. Virology 462–463:49–59

32. Salsman J, Top D, Boutilier J, Duncan R (2005) Extensive syn-

cytium formation mediated by the reovirus FAST proteins trig-

gers apoptosis-induced membrane instability. J Virol

79:8090–8100

33. Schnitzer TJ, Ramos T, Gouvea V (1982) Avian reovirus

polypeptides: analysis of intracellular virus-specified products,

virions, top component, and cores. J Virol 43:1006–1014

34. Shih WL, Hsu HW, Liao MH, Lee LH, Liu HJ (2004) Avian

reovirus sigmaC protein induces apoptosis in cultured cells.

Virology 321:65–74

35. Shmulevitz M, Yameen Z, Dawe S, Shou J, O’Hara D, Holmes I,

Duncan R (2002) Sequential partially overlapping gene

arrangement in the tricistronic S1 genome segments of avian

reovirus and Nelson Bay reovirus: implications for translation

initiation. J Virol 76:609–618

36. Shmulevitz M, Salsman J, Duncan R (2003) Palmitoylation,

membrane-proximal basic residues, and transmembrane glycine

residues in the reovirus p10 protein are essential for syncytium

formation. J Virol 77:9769–9779

37. Shmulevitz M, Corcoran J, Salsman J, Duncan R (2004) Cell–cell

fusion induced by the avian reovirus membrane fusion protein is

regulated by protein degradation. J Virol 78:5996–6004

38. Top D, Barry C, Racine T, Ellis CL, Duncan R (2009) Enhanced

fusion pore expansion mediated by the trans-acting Endodomain

of the reovirus FAST proteins. PLoS Pathog 5:e1000331

39. Varela R, Benavente J (1994) Protein coding assignment of avian

reovirus strain S1133. J Virol 68:6775–6777

40. Varela R, Martinez-Costas J, Mallo M, Benavente J (1996)

Intracellular posttranslational modifications of S1133 avian reo-

virus proteins. J Virol 70:2974–2981

41. Zhang Z, Lin W, Li X, Cao H, Wang Y, Zheng SJ (2015) Critical

role of eukaryotic elongation factor 1 alpha 1 (EEF1A1) in avian

reovirus sigma-C-induced apoptosis and inhibition of viral

growth. Arch Virol 160:1449–1461

Role of LAMP-1 in avian reovirus P10 degradation 911

123

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具