Journal of General Virology (2014), 95, 1211–1220

DOI 10.1099/vir.0.063511-0

Palmitoylation is required for intracellular trafficking of influenza B virus NB protein and efficient influenza B virus growth in vitro Andrew Demers,1,2,33 Zhiguang Ran,2,33 Qiji Deng,2,3 Dan Wang,1,3 Brody Edman,1,3 Wuxun Lu1,3 and Feng Li1,2,3 Correspondence Feng Li [email protected]

1

Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA

2

Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD 57007, USA

3

Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings, SD 57007, USA

Received 8 January 2014 Accepted 24 March 2014

All influenza viruses bud and egress from lipid rafts within the apical plasma membrane of infected epithelial cells. As a result, all components of progeny virions must be transported to these lipid rafts for assembly and budding. Although the mechanism of transport for other influenza proteins has been elucidated, influenza B virus (IBV) glycoprotein NB subcellular localization and transport are not understood completely. To address the aforementioned properties of NB, a series of trafficking experiments were conducted. Here, we showed that NB co-localized with markers specific for the endoplasmic reticulum (ER) and Golgi region. The data from chemical treatment of NB-expressing cells by Brefeldin A, a fungal antibiotic and a known chemical inhibitor of the protein secretory pathway, further confirmed that NB is transported through the ER–Golgi pathway as it restricted NB localization to the perinuclear region. Using NB deletion mutants, the hydrophobic transmembrane domain was identified as being required for NB transport to the plasma membrane. Furthermore, palmitoylation was also required for transport of NB to the plasma membrane. Systematic mutation of cysteines to serines in NB demonstrated that cysteine 49, likely in a palmitoylated form, is also required for transport to the plasma membrane. Surprisingly, further analysis demonstrated that in vitro replication of NBC49S mutant virus was delayed relative to the parental IBV. The results demonstrated that NB is the third influenza virus protein to have been shown to be palmitoylated and together these findings may aid in future studies aimed at elucidating the function of NB.

INTRODUCTION Influenza B virus (IBV) is an important respiratory pathogen that co-circulates with influenza A virus (IVA) (family Orthomyxoviridae; genus Influenzavirus B and Influenzavirus A, respectively) and contributes to influenza-related morbidity and mortality in humans (Palese & Shaw, 2007). IBV is an enveloped virus containing eight negative-sense RNA segments. RNA segment 6 of the IBV genome encodes two proteins; neuraminidase (NA) and a 100 aa glycoprotein designated NB, which is exclusive to IBV (Shaw et al., 1983). The function of NB remains unknown; however, a study by Hatta and Kawaoka (2003) showed that NB is dispensable for virus replication in vitro (Hatta & Kawaoka, 2003). They also found that NB 3These authors contributed equally to this work. One supplementary figure is available with the online version of this paper.

063511 G 2014 The Authors

Printed in Great Britain

knockout virus was attenuated in mice (Hatta & Kawaoka, 2003). As NB is highly conserved among IBVs, it is likely NB has a functional role in vivo (Hatta & Kawaoka, 2003). NB is a type III integral membrane protein that transverses the membrane once, and contains an 18 aa N-terminal ectodomain, a 22 aa hydrophobic transmembrane domain and a 60 aa cytoplasmic tail (Shaw et al., 1983; Williams & Lamb, 1986). IBV assembles and buds at the apical portion of the plasma membrane of infected cells (Rodriguez Boulan & Pendergast, 1980; Rodriguez Boulan & Sabatini, 1978; Roth et al., 1983). As a result of this budding location, all structural and non-structural proteins as well as RNA segments must be brought to the assembly site in the apical plasma membrane (Barman et al., 2001). NB is expressed abundantly at the cell surface (Williams & Lamb, 1986) and is also a component of the virion, with 15–100 molecules being incorporated per virion (Brassard et al., 1996). Although NB is transported to the plasma 1211

A. Demers and others

membrane, the transport mechanism is unknown. A study by Williams and Lamb (1988) showed that NB glycosylation is not required for NB transport to the plasma membrane, although it is modified by N-glycosylation – a process that occurs in the endoplasmic reticulum (ER) (Williams & Lamb, 1988). This suggests that NB is likely co-translated in the ER; however, NB lacks a cleavable Nterminal signal sequence (Williams & Lamb, 1986) common among proteins that are co-translated in the ER and subsequently transported through the Golgi en route to the plasma membrane (Osborne et al., 2005). Although the NB membrane-targeting signal is undetermined, it has been suggested that the transmembrane domain of NB acts as both a targeting signal for transport and an anchor for integration into the plasma membrane (Williams & Lamb, 1986). In comparison, IBV proteins NA and haemagglutinin (HA) contain characterized determinants for membrane trafficking in their transmembrane domains (Kundu et al., 1996; Lin et al., 1998). Among the determinants in HA are three critical cysteine residues located in the boundary region between the transmembrane and cytoplasmic tail domains. These cysteines undergo post-translational modification by the addition of palmitic acid (palmitoylation or S-acylation) through a thioether linkage (Veit et al., 1991). Studies have demonstrated that palmitoylation of these cysteines is not critical for transport of HA; however, they may play a role in HA-mediated membrane fusion. Studies examining the role of palmitoylation of HA have produced conflicting results; this may be due to the dynamic process of palmitoylation modification of proteins (Naeve & Williams, 1990; Simpson & Lamb, 1992; Steinhauer et al., 1991). NB has been shown to be palmitoylated; however, which cysteine residues act as palmitoylation acceptors and the role of palmitoylation of NB remain unknown (Brassard et al., 1996). Several cellular proteins have been shown to require palmitoylation for targeting to lipid raft domains within plasma membranes, such as B-cell tetraspanin CD81 (Cherukuri et al., 2004) and several proteins of the Src kinase family (Koegl et al., 1994). It is possible that palmitoylation could play a role in the transport of NB to the plasma membrane, despite the fact that it does not play a role in the transport of other influenza proteins to the plasma membrane. In this study, we demonstrated that NB localizes in the perinuclear region and utilizes the ER–Golgi secretory pathway and palmitoylation of cysteine 49 for efficient transport to the plasma membrane. Furthermore, we showed that the growth kinetics of a mutant virus with disruption of palmitoylation of cysteine 49 of NB was delayed relative to WT IBV in cell culture.

RESULTS Subcellular localization of IBV NB Native antibody specific to IBV NB was not available at the time of this study, so a construct in which an epitope tag 1212

was fused to the C terminus of NB was generated. The NB fusion constructs were detected with detergent treatment followed by immunofluorescent staining. COS-1 cells transfected with NB fusion constructs displayed a staining pattern at the plasma membrane indicating that the protein was localized at the plasma membrane (data not shown). The data indicate that fusion of an HA or Myc epitope tag to the C terminus did not alter NB localization as an integral membrane protein. This result could also be seen when a Golgi marker was used (Fig. 1b). The results of our experiment indicate that the NB subcellular localization is in the ER–Golgi/perinuclear region. Two well-characterized markers for labelling ER and Golgi compartments, i.e. ERGIC-53 (ER–Golgi intermediate compartment) and Giantin (Golgi apparatus), were also used in this study. Our data showed that NB likely resides in the ER–Golgi region and is transported to the plasma membrane (Fig. 1a). To confirm this hypothesis, a protein co-localization experiment was performed. Fluorescent signals of NB were seen both in the perinuclear region and in the plasma membrane (Fig. 1a), similar to what was observed for the subcellular localization pattern displayed by the pNB-HA fusion protein. Also, NB co-localized with ERGIC-53, as indicated by the bright fluorescence observed in the perinuclear region (white arrows in the merged panel indicate co-localization). This indicated that NB is transported to this intermediate compartment. To demonstrate that NB is transported to the Golgi, we used an anti-Giantin antibody to label the Golgi apparatus. Fluorescence signals for NB were similar to what was seen previously (Fig. 1a), and co-localization was also observed for NB and the Giantin marker (white arrows in the merged panel indicate co-localization). Fig. 1(a, b) confirmed that NB is transported from the ER in an ER–Golgi compartment to the Golgi apparatus and ultimately to the plasma membrane. To validate the above observation of protein localization and confirm precisely if NB was transported to the plasma membrane through the ER–Golgi secretory pathway, cells expressing pNB-HA were incubated in the presence or absence of Brefeldin A (BFA; 5 mg ml21), a fungal antibiotic and a known inhibitor of the ER–Golgi secretory pathway. BFA blocks the transport of proteins through the ER–Golgi to the plasma membrane by disrupting anterograde protein transport from the ER to the Golgi apparatus (Fujiwara et al., 1988). In BFA-treated cells, NB was located in the perinuclear region, not the plasma membrane (Fig. 1c, merged panel). Restricted localization of NB in the perinuclear region by BFA treatment confirmed that NB utilized the ER–Golgi secretory pathway to reach the cell surface. Identification of IBV NB membrane-targeting signal To identify the protein domain responsible for NB trafficking to the cell membrane, a series of truncation mutants of NB were generated in which each of the Journal of General Virology 95

Intracellular trafficking of IBV NB protein

(a)

NB-HA

ERGIC-53

DAPI

Merge

(b)

NB-HA

Giantin

DAPI

Merge

(c)

FITC

DAPI

Merge

BFA treatment

Fig. 1. Cellular co-localization and intracellular trafficking of IBV NB. (a) Cellular co-localization of NB with ERGIC-53 ER– Golgi intermediate compartments. COS-1 cells transfected transiently with pNB-HA were fixed, permeabilized with Triton X100, and stained with DAPI (blue), an anti-HA mAb (He et al., 2003) and antibody against ERGIC-53 (red). (b) Cellular colocalization of NB with the Golgi apparatus. COS-1 cells transfected transiently with pNB-HA were fixed, permeabilized with Triton X-100, and stained with DAPI (blue), an anti-HA mAb (He et al., 2003) and antibody against Giantin (a Golgi marker) (red). Merged profiles are shown (Merge) and white arrows indicate co-localization. (c) Characterization of the inhibitory effect on NB transport by BFA. COS-1 cells grown on glass coverslips were transfected with pNB-HA plasmids in the presence or absence of inhibitory BFA (5 mg ml”1). Cells were fixed with formaldehyde at 24 h post-transfection, permeabilized with Triton X-100 and immunostained with anti-HA mAb.

structural domains of NB was deleted in a systematic fashion. The localization of NB deletion mutants in transfected cells should allow for the identification of regions required for cellular transport. We focused our mutagenesis and construct generation on transmembrane and intracellular cytoplasmic domains of NB because numerous studies have shown these regions are responsible for protein sorting and targeting to the membranes for other proteins (Guerriero et al., 2008). To this end, a panel of deletion mutants spanning a portion of the N terminus, the entire transmembrane domain or the cytoplasmic domain of NB was generated (Fig. 2a). The mutants NBD10–18 and NBD81–100 showed a localization pattern similar to that of WT NB (Fig. 2b), indicating that aa 10–18 and 81–100 were not required for cellular transport. Note that a deletion mutant spanning aa 2–18 was also made; however, for an unknown reason, this construct did not express in transfected cells. http://vir.sgmjournals.org

Conversely, mutants NBD19–40 and NBD41–80 showed a divergent localization pattern from that of WT NB (Fig. 2b). Over 50 % (Table 1) of the cells expressing mutant NBD19–40 accumulated predominantly in the nucleus and perinuclear region, and showed less membrane staining, whilst 67 % (Table 1) of the cells expressing mutant NBD41–80 localized in the perinuclear region with no membrane staining. These results indicated that the transmembrane domain of NB and aa 41–80 of NB are required for cellular transport of NB to the plasma membrane. Cysteine 49 in IBV NB is required for transport of NB to the plasma membrane Palmitoylation of M2 and HA of IVA virus has been shown to be important for cellular transport of these proteins. Palmitoylation occurs at cysteine residues within proteins 1213

A. Demers and others

TRITC

MERGE

DAPI

(b)

(a) N

NB/Myc

N-terminal ectodomain aa 1–18 Transmembrane domain aa 19–40

Plasma membrane Predicted helix 1 aa 41–80

Cytoplasmic tail domain aa 41–100 C

NBD10–18

Predicted helix 2 aa 81–100 NBD19–40

NBD10–18 N

C

NBD19–40 N

C

NBD41–80

NBD41–80 N

C

NBD81–100 N

C

NBD81–100

Fig. 2. Intracellular localization of NB truncation mutants in transfected cells. (a) Diagram of NB structural domains and schematic diagram of expression of plasmids encoding NB deletion mutants. Black segments represent the defined deletions of NB protein as indicated. (b) Intracellular localization of NB mutants. COS-1 cells were transfected with pNB-Myc or NB deletion mutants, fixed at 48 h post-transfection and stained with anti-c-Myc mAb (red).

and therefore we chose to first examine the effect of mutating systematically these cysteines in NB. There are seven cysteines in NB with one in the N-terminal ectodomain, two in the hydrophobic transmembrane domain and four in the first predicted helix (aa 41–80) in the cytoplasmic tail domain (Fig. 3a). A series of NB

site-directed mutants were generated where selected cysteines were mutated to serines (Fig. 3a). All the mutants had a pattern similar to WT except for NBC49S, in which 70 % (Table 2) of the cells displayed a fluorescent pattern restricted to the perinuclear region only (Fig. 3b), similar to that seen for the truncation mutant NBD41–80 (67 %;

Table 1. Cellular localization of NB truncation mutants Mutant NB/Myc NBD10–18 NBD19–40 NBD41–80 NBD81–100

1214

Total cells positive for TRITC (N)

Plasma membrane +perinuclear region [n (%)]

Nucleus [n (%)]

72 24 66 49 52

20 (28) 12.5 (52) 10 (15) 0 12 (23)

0 0 34 (52) 16 (33) 5 (10)

Perinuclear region only [n (%)] 52 11.5 22 33 35

(72) (48) (33) (67) (67)

Journal of General Virology 95

Intracellular trafficking of IBV NB protein

TRITC

DAPI

Merge

(b)

(a) N-terminal ectodomain

NBC8S

NBC49S

NBC62S First predicted helix in cytoplasmic tail domain

NBC65S

NBC69S

Fig. 3. Localization of NB cysteine-to-serine mutants. (a) Schematic displaying structural domains of NB and positions of cysteines in NB. (b) Localization of cysteine-to-serine mutants. Cells were transfected with NB cysteine-to-serine mutant plasmid DNA. At 48 h post-transfection, cells were fixed in 4 % formaldehyde and stained with anti-c-Myc conjugated to TRITC.

Table 1) (Fig. 2b). These data indicated that cysteine at position 49 in the first predicted helix of NB is required for the transport of NB to the plasma membrane and provides an explanation for why NBD41–80 has a restricted localization in Fig. 2b. IBV NB transport to the plasma membrane requires palmitoylation To determine the effect of palmitoylation on the transport of NB to the plasma membrane we utilized a pharmacological inhibitor of protein palmitoylation, called 2-bromopalmitate (2BP) (Webb et al., 2000). The majority of pNB-HA transfected cells (69 %; Table 3) treated with 2BP displayed a fluorescent pattern restricted to the perinuclear region with no plasma membrane staining observed (Fig. 4) – a pattern similar to that seen when aa 41–80 of NB were deleted (Fig. 2b). This indicated that palmitoylation is required for the transport of NB to the plasma membrane. Palmitoylation of IBV NB is required for efficient growth of IBV in vitro Next, we evaluated the effect of NBC49S mutation on IBV replication by comparing the replication kinetics of http://vir.sgmjournals.org

parental IBV and NBC49S mutant virus stocks in cultures of MDCK cells. Virus stocks collected from transfected 293T/MDCK co-cultures with corresponding IBV reversegenetics systems were analysed for their infectivity (TCID50) in MDCK cells. Fresh MDCK cells were then infected in parallel with either the WT or the NBC49S mutant at an m.o.i. of 0.01. Aliquots of the tissue culture supernatant were taken at periodic intervals and analysed by using the standard HA assay for the measurement of virus replication kinetics. Although no significant differences could be observed in peak viral titres of the viruses after 60 h, the initial replication of the NBC49S mutant viruses was delayed. For example, at 36 h post-infection, NBC49S mutant viruses had an HA titre 2 log2 lower than that of WT virus (Fig. 5). The effect was less pronounced at 48 h post-infection where only 1 log2 difference was observed between WT and mutant viruses. The NA enzymic activity assay performed at 32 h post-infection revealed no differences between rYam98 WT and NBC49S viruses, indicating that the delayed growth kinetics observed was not attributable to a defect in NA (Fig. S1, available in the online Supplementary Material). These data indicated that the effect of NBC49S mutation did not affect overall virus growth, but rather delayed growth at early time points. 1215

A. Demers and others

Table 2. Cellular localization of NB cysteine-to-serine mutants Mutant

Total cells positive for TRITC (N)

NBC8S NBC49S NBC62S NBC65S NBC69S

82 53 58.5 65.5 76

Plasma membrane +perinuclear region [n (%)] 47 16 44 49 49

(57) (30) (75) (75) (65)

DISCUSSION IBV glycoprotein NB is a type III integral membrane protein that lacks an N-terminal leader signal sequence and is modified by N-glycans in the lumen of the ER. Here, we demonstrated that NB is transported through the ER–Golgi intermediate pathway to the plasma membrane utilizing its hydrophobic transmembrane domain presumably for integration into the ER membrane and palmitoylation of cysteine at position 49 in the cytoplasmic tail domain. Furthermore, both palmitoylation of cysteine 49 and an intact transmembrane domain of NB are required for the transport of NB to the plasma membrane. We also demonstrated that NB co-localized with both Golgi and ER–Golgi intermediate specific markers. NB localization was restricted to the perinuclear region in the presence of a known chemical inhibitor of the ER-Golgi protein secretory pathway, BFA. From these results we conclude that NB is transported to the plasma membrane via the ER–Golgi secretory pathway. This finding confirmed a previous hypothesis that NB used the ER–Golgi secretory pathway and is not surprising as NB is modified by N-glycans – a process that occurs in the lumen of the ER (Williams & Lamb, 1986). Furthermore, this demonstrated that the route of transport for NB is the same as for the other IBV envelope proteins M2, HA and NA (Watanabe et al., 2003). Although NB has been shown to be dispensable for replication in vitro, it does play a role in viral fitness (Hatta & Kawaoka, 2003) in vivo, possibly through increasing viral replication efficiency or modulation of host viral immune responses. For the previous assumption, a common trafficking pathway for IBV envelope proteins serves to provide an environment that could facilitate necessary protein–protein interactions, promoting the production of a virus with improved fitness in the host. We also found that the transmembrane domain of NB serves as a membrane-targeting signal. Although the exact

Nucleus [n (%)] 1 (1) 0 0 0 1 (1)

Perinuclear region only [n (%)] 34 37 14.5 16.5 26

(42) (70) (25) (25) (34)

mechanism by which the transmembrane domain serves as a localization signal is not known, it is likely that loss of the transmembrane domain prevents the incorporation of NB into the ER membrane, as it has been shown that NB lacks an N-terminal cleavable signal sequence (Williams & Lamb, 1986) – a sequence common among proteins destined for transport through the ER. It remains unclear how exactly the transmembrane domain is responsible for the incorporation of NB in the ER membrane. The NB deletion mutant lacking the transmembrane domain (NBD19–40) showed no localization at the plasma membrane, but rather was found in the nucleus (Fig. 2b). NB lacks any apparent nuclear localization signals, so it is likely that the protein diffused passively into the nucleus, as NB mutants lacking the transmembrane domain would be significantly smaller than the 40 kDa molecular mass limitation for passive diffusion through the nuclear pores (Terry et al., 2007). The abundance of NB in the nucleus may also be explained by the overall positive charge of NB under physiological conditions (pI 8.5), which may allow NB to interact with the negatively charged DNA and could explain why so much NB is apparently trapped within the nucleus. There are a total of seven cysteines in NB: one in the Nterminal ectodomain, two in the hydrophobic transmembrane domain and four in the first helix of the cytoplasmic tail domain. Due to the fact that NBD41–80 had restricted localization and it contains four out of the seven cysteines in the protein sequence of NB, we decided to examine the role of palmitoylation in the transport of NB to the plasma membrane. Palmitoylation is a frequent post-translational modification of proteins in which a 16-carbon fatty acid called palmitate is added to internal cysteine residues. Palmitoylation has been shown to contribute to membrane association, protein sorting and stability of proteins.

Table 3. Cellular localization of NB in the presence of palmitoylation inhibitor 2BP Mutant NB/HA NB/HA+2BP

1216

Total cells positive for TRITC (N)

Plasma membrane +perinuclear region [n (%)]

Nucleus [n (%)]

Perinuclear region only [n (%)]

167 147

135 (81) 46 (31)

0 0

32 (19) 101 (69)

Journal of General Virology 95

Intracellular trafficking of IBV NB protein

FITC

DAPI

Merge

NB-HA Untreated

NB-HA +2BP

Fig. 4. Localization of NB-HA in the presence of palmitoylation inhibitor 2BP. Cells were transfected with pNB-HA plasmid DNA. At 4 h post-transfection, cell culture medium was changed with either fresh medium (untreated) or fresh medium containing 100 mM 2BP. At 48 h post-transfection, cells were fixed in 4 % formaldehyde and stained with anti-HA conjugated to FITC. In the presence of palmitoylation inhibitor 2BP fluorescence is restricted to the perinuclear region, whereas untreated cells show a pattern indicative of the plasma membrane.

Mean HA titre (log2)

Palmitoylation is a readily reversible process which may regulate the functional activities of integral membrane proteins (Bijlmakers & Marsh, 2003). This is applicable to NB because palmitoylation has also been shown to play an important role in the targeting of proteins to lipid rafts (Melkonian et al., 1999). Lipid raft microdomains are dynamic structures in cellular membranes that are rich in cholesterol and sphingolipids, and can serve as platforms for signal transduction as well as protein incorporation (Simons & Toomre, 2000). Several enveloped viruses, including influenza viruses, utilize these lipid rafts for budding and assembly from infected cells (Scheiffele et al.,

8 7 6 5 4 3 2 1 0

0

12

24 36 48 60 Time (h post-infection)

72

Fig. 5. Growth kinetics of WT and NBC49S mutant viruses in MDCK cells. MDCK cells grown in six-well plates were infected with either parental IBV (&) or the NBC49S mutant virus ($) at an m.o.i. of 0.01. Virus replication was monitored at various intervals following infection by measuring the HA titre in the culture supernatant, which was expressed as the log2 reciprocal titre. The data shown represent the mean±SD of two independent experiments. http://vir.sgmjournals.org

1999; Takeda et al., 2003). Palmitoylation of NB has been reported previously; however, the specific cysteine residues that undergo palmitoylation have not been identified (Brassard et al., 1996). Localization of all but a single cysteine mutant showed a pattern similar to that of WT NB. Localization of the NBC49S mutant was restricted to the perinuclear region, displaying no localization at the plasma membrane. These data demonstrated that cysteine 49 in the first predicted helix of the cytoplasmic tail domain of NB is required for the transport of NB to the plasma membrane. WT NB in the presence of 2BP displayed a localization pattern restricted to the perinuclear region, similar to what is observed for NBD41–80 or NBC49S mutants, indicating cysteine at position 49 is likely palmitoylated. Several viral and cellular integral membrane proteins are palmitoylated on cysteines that are either close to their transmembrane/cytoplasmic tail domain boundary region or located in their cytoplasmic tail domain (Bijlmakers & Marsh, 2003). Both IVA HA and M2 have cysteines that undergo palmitoylation located in their transmembrane domain/cytoplasmic tail domain boundary regions; however, palmitoylation of these cysteines is not required for their transport to the plasma membrane (Holsinger et al., 1995; Veit et al., 1991). Cysteine 49 is located 9 aa from the transmembrane/cytoplasmic tail domain boundary region. Some cellular transmembrane proteins have been shown to require palmitoylation for transport to the plasma membrane, such as chemokine receptor CCR5 (Blanpain et al., 2001; Kraft et al., 2001). IAV M2 has a single palmitoylation site, which does not require palmitoylation for virus replication; however, it does affect viral virulence in mice (Grantham et al., 2009). 1217

A. Demers and others

NBC49S mutant virus had significantly delayed growth kinetics in vitro compared with WT, which suggests that efficient trafficking of NB to the surface is required for efficient growth of the virus in vitro, but this effect may also be true in vivo given the effect observed in the case of IVA M2 which is structurally analogous to NB. Although the reason for NBC49S virus delayed growth kinetics is unclear, it is possible that an accumulation of NB in the ER could induce either transient or permanent interactions between NB and other membrane proteins, such as M2, HA or NA. Along these lines, the HA titre could be lowered if HA is being sequestered within the cell. Analysis of 3994 (all available sequences at the date of this publication) NB amino acid sequences from the NCBI Influenza Virus Resource demonstrated that 3967 (99 %) sequences contained a cysteine at position 49, indicating this cysteine is highly conserved at this position. This level of conservation likely indicates this position is important for NB function and/or virus infectivity (replication or virulence). Further studies are needed to elucidate the effect of NBC49S both in vitro and in vivo on virus growth and pathogenesis. In summary, NB is transported through the ER–Golgi secretory pathway utilizing two important signals: the hydrophobic transmembrane domain and palmitoylation of cysteine 49 in the cytoplasmic tail domain of NB. Furthermore, palmitoylation of cysteine 49 is necessary for efficient IBV growth in vitro. These results may serve as valuable information in studies aimed at elucidating the role of NB in the replication, transmission and pathogenesis of IBV.

METHODS Plasmids. PCR and cloning were performed with proofreading Vent

DNA polymerase (New England Biolabs) under standard reaction conditions. The integrity of all PCR-derived DNA fragments was verified by sequencing. For expression of viral protein NB used in the study, the cDNA sequence encoding NB was amplified individually from B/Lee/40 (Dauber et al., 2004) by PCR and subcloned into eukaryotic expression vector pCAGGS (Matsuda & Cepko, 2004). For construction of NBD10–18, NBD19–40, NBD41–80 and NBD81–100 mutants, overlapping PCR in conjunction with primers excluding specific regions of NB was used. Cysteine-to-serine point mutants were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and phosphorylated primers (sequences available from the authors upon request). Cells, transfection and antibodies. COS-1 and MDCK cells were

cultured in Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10 % FBS. mAb against the HA epitope was purchased from Sigma-Aldrich. mAb against c-Myc epitope conjugated to tetramethylrhodamine (TRITC) was purchased from Santa Cruz Biotechnology. Golgi apparatus staining was performed using polyclonal antibody against Giantin purchased from Covance, followed by incubation with anti-rabbit IgG conjugated to Alexa Fluor 594 (Invitrogen). Staining of the ER–Golgi intermediate was performed using rabbit anti-ERGIC-53/p58 conjugated to Cy3 1218

(Sigma-Aldrich). For all transfections, cells were transfected with 1 mg DNA using TransIT-LT1 transfection reagent (Mirus Bio) following the manufacturer’s protocol. Immunofluorescence assay and confocal microscopy. We

conducted immunostaining using the anti-Giantin antibody specific to Giantin, a Golgi-resident protein, coupled with anti-rabbit IgG conjugated to Alexa Fluor 594 and ER–Golgi intermediate vesicles, anti-ERGIC53/p58 conjugated to Cy3. We also used the wellcharacterized chemical inhibitor BFA to block protein transport through the ER–Golgi to the plasma membrane. BFA inhibits normal cellular protein transport functions by disruption of anterograde protein transport from the ER to the Golgi apparatus (Fujiwara et al., 1988). COS-1 cells were transfected transiently with the pNB-HA expression plasmid and fixed at 48 h post-transfection. Cells were then stained with anti-HA conjugated to FITC and anti-ERGIC53/p58 or antiGiantin. COS-1 cells grown on coverslips were transfected with NB expression plasmids (pNB-HA and pNB-Myc). At 24 or 48 h after transfection, cells were washed and fixed with 4 % formaldehyde (Electron Microscopy Sciences). Cells were then permeabilized with 0.2 % (v/v) Triton X-100 in PBS. Cells were blocked with 2 % BSA in PBS prior to incubation with the appropriate primary antibody conjugated directly with either anti-HA conjugated with FITC or anti-C-Myc conjugated with TRITC. Cells were finally washed briefly in 0.5 % BSA in PBS before being mounted on a slide with ProLong Gold antifade reagent containing DAPI dye (Invitrogen), which was used to stain the nuclei. Fluorescent imaging of fixed cells was done using a FluoView FV300 confocal system (Olympus) equipped with an IX81 microscope. Digital images were processed with Adobe Photoshop (version 6). All images were taken under similar experimental conditions (i.e. exposure time, magnification and intensification) and were subject to standardized processing. For protein co-localization assays, cells expressing the pNB-HA protein were fixed and permeabilized with Triton X-100 prior to incubation with combinations of primary antibodies and Alexa Fluor 594-conjugated goat secondary antibody. For the pNB transport inhibition experiment, 5 mg BFA ml21 (Sigma-Aldrich) was added to COS-1 cells 24 h post-transfection. BFA was maintained in cell culture throughout the experiment, which lasted 24 h on average. A similar procedure as described above was used to visualize either the pNB-HA protein or cellular markers (ERGIC-53 and Giantin). Palmitoylation inhibitor treatment. A pharmacological inhibitor

of protein palmitoylation, 2BP (Sigma-Aldrich), was used to address the effect of palmitoylation on transport of palmitoylated NB to the cell surface. COS-1 cells were transfected with pNB-HA as described above. At 4 h post-transfection, 100 mM 2BP was added and maintained throughout the period of the culture. Immunofluorescence assay using anti-HA/FITC antibody was performed. Virus growth kinetics and haemagglutination assay. The

protocol used for rescue of the parental IBV or NB C49S mutant virus was based on the protocol for generation of IBV (McCullers et al., 2005). The haemagglutination assay was conducted following the WHO standard procedure. MDCK cells were infected in parallel with either the WT or the NBC49S mutant at an m.o.i. of 0.01. Aliquots of the tissue culture supernatant were taken at periodic intervals and analysed by using the haemagglutination assay for the measurement of virus replication kinetics. Virus replication experiments were conducted using two independent experiments, each performed in triplicate. Journal of General Virology 95

Intracellular trafficking of IBV NB protein

NA activity assay. NA activity was measured by using a standard fluorescent assay. The clarified supernatant and fluorogenic substrate MUNANA (Sigma) were added to a 96-well plate, mixed, and incubated at 37 uC for 1 h. After stopping the reaction by adding 50 mM glycine buffer, fluorescence was measured in a fluorometer with an excitation wavelength of 355 nm and an emission wavelength of 460 nm, and relative fluorescent units were recorded.

ACKNOWLEDGEMENTS We thank Elizabeth Kolb for editing this manuscript. Research was supported by the South Dakota State University AES Fund (3AH203 to F. L.) and NIH Public Health Service grants (AI0781775 and K02AI076125-02) to F. L.

Kraft, K., Olbrich, H., Majoul, I., Mack, M., Proudfoot, A. & Oppermann, M. (2001). Characterization of sequence determinants

within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J Biol Chem 276, 34408–34418. Kundu, A., Avalos, R. T., Sanderson, C. M. & Nayak, D. P. (1996).

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. J Virol 70, 6508–6515. Lin, S., Naim, H. Y., Rodriguez, A. C. & Roth, M. G. (1998). Mutations

in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells. J Cell Biol 142, 51–57. Matsuda, T. & Cepko, C. L. (2004). Electroporation and RNA

interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A 101, 16–22.

REFERENCES

McCullers, J. A., Hoffmann, E., Huber, V. C. & Nickerson, A. D. (2005). A single amino acid change in the C-terminal domain of the

Barman, S., Ali, A., Hui, E. K., Adhikary, L. & Nayak, D. P. (2001).

matrix protein M1 of influenza B virus confers mouse adaptation and virulence. Virology 336, 318–326.

Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses. Virus Res 77, 61–69. Bijlmakers, M. J. & Marsh, M. (2003). The on-off story of protein

palmitoylation. Trends Cell Biol 13, 32–42.

Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G. & Brown, D. A. (1999). Role of lipid modifications in targeting proteins to

detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem 274, 3910–3917.

Blanpain, C., Wittamer, V., Vanderwinden, J. M., Boom, A., Renneboog, B., Lee, B., Le Poul, E., El Asmar, L., Govaerts, C. & other authors (2001). Palmitoylation of CCR5 is critical for receptor

Naeve, C. W. & Williams, D. (1990). Fatty acids on the A/Japan/305/57

trafficking and efficient activation of intracellular signaling pathways. J Biol Chem 276, 23795–23804.

Osborne, A. R., Rapoport, T. A. & van den Berg, B. (2005). Protein

influenza virus hemagglutinin have a role in membrane fusion. EMBO J 9, 3857–3866.

Brassard, D. L., Leser, G. P. & Lamb, R. A. (1996). Influenza B virus

translocation by the Sec61/SecY channel. Annu Rev Cell Dev Biol 21, 529–550.

NB glycoprotein is a component of the virion. Virology 220, 350–360.

Palese, P. & Shaw, M. L. (2007). In Fields Virology, 5th edn, pp. 1647–

Cherukuri, A., Carter, R. H., Brooks, S., Bornmann, W., Finn, R., Dowd, C. S. & Pierce, S. K. (2004). B cell signaling is regulated by

1689. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Williams & Wilkins.

induced palmitoylation of CD81. J Biol Chem 279, 31973–31982.

Rodriguez Boulan, E. & Pendergast, M. (1980). Polarized distri-

Dauber, B., Heins, G. & Wolff, T. (2004). The influenza B virus

bution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell 20, 45–54.

nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction. J Virol 78, 1865–1872. Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A. & Ikehara, Y. (1988).

Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem 263, 18545–18552.

Rodriguez Boulan, E. & Sabatini, D. D. (1978). Asymmetric budding

of viruses in epithelial monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci U S A 75, 5071–5075. Roth, M. G., Srinivas, R. V. & Compans, R. W. (1983). Basolateral

maturation of retroviruses in polarized epithelial cells. J Virol 45, 1065– 1073.

Grantham, M. L., Wu, W. H., Lalime, E. N., Lorenzo, M. E., Klein, S. L. & Pekosz, A. (2009). Palmitoylation of the influenza A virus M2 protein

Scheiffele, P., Rietveld, A., Wilk, T. & Simons, K. (1999). Influenza

is not required for virus replication in vitro but contributes to virus virulence. J Virol 83, 8655–8661.

viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 274, 2038–2044.

Guerriero, C. J., Lai, Y. & Weisz, O. A. (2008). Differential sorting and

Shaw, M. W., Choppin, P. W. & Lamb, R. A. (1983). A previously

Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway. J Biol Chem 283, 18040–18047.

unrecognized influenza B virus glycoprotein from a bicistronic mRNA that also encodes the viral neuraminidase. Proc Natl Acad Sci U S A 80, 4879–4883.

Hatta, M. & Kawaoka, Y. (2003). The NB protein of influenza B virus

Simons, K. & Toomre, D. (2000). Lipid rafts and signal transduction.

is not necessary for virus replication in vitro. J Virol 77, 6050–6054.

Nat Rev Mol Cell Biol 1, 31–39.

He, X. S., Mahmood, K., Maecker, H. T., Holmes, T. H., Kemble, G. W., Arvin, A. M. & Greenberg, H. B. (2003). Analysis of the frequencies

Simpson, D. A. & Lamb, R. A. (1992). Alterations to influenza virus

and of the memory T cell phenotypes of human CD8+ T cells specific for influenza A viruses. J Infect Dis 187, 1075–1084.

hemagglutinin cytoplasmic tail modulate virus infectivity. J Virol 66, 790–803. Steinhauer, D. A., Wharton, S. A., Wiley, D. C. & Skehel, J. J. (1991). Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has

Holsinger, L. J., Shaughnessy, M. A., Micko, A., Pinto, L. H. & Lamb, R. A. (1995). Analysis of the posttranslational modifications of the

no effect on membrane fusion properties. Virology 184, 445–448.

influenza virus M2 protein. J Virol 69, 1219–1225.

Takeda, M., Leser, G. P., Russell, C. J. & Lamb, R. A. (2003).

Koegl, M., Zlatkine, P., Ley, S. C., Courtneidge, S. A. & Magee, A. I. (1994). Palmitoylation of multiple Src-family kinases at a homolog-

Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc Natl Acad Sci U S A 100, 14610– 14617.

ous N-terminal motif. Biochem J 303, 749–753. http://vir.sgmjournals.org

1219

A. Demers and others Terry, L. J., Shows, E. B. & Wente, S. R. (2007). Crossing the nuclear

Webb, Y., Hermida-Matsumoto, L. & Resh, M. D. (2000). Inhibition

envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318, 1412–1416.

of protein palmitoylation, raft localization, and T cell signaling by 2bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275, 261–270.

Veit, M., Kretzschmar, E., Kuroda, K., Garten, W., Schmidt, M. F. G., Klenk, H.-D. & Rott, R. (1991). Site-specific muta-

genesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. J Virol 65, 2491– 2500. Watanabe, S., Imai, M., Ohara, Y. & Odagiri, T. (2003). Influ-

enza B virus BM2 protein is transported through the trans-Golgi network as an integral membrane protein. J Virol 77, 10630– 10637.

1220

Williams, M. A. & Lamb, R. A. (1986). Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region. Mol Cell Biol 6, 4317–4328. Williams, M. A. & Lamb, R. A. (1988). Polylactosaminoglycan

modification of a small integral membrane glycoprotein, influenza B virus NB. Mol Cell Biol 8, 1186–1196.

Journal of General Virology 95

Palmitoylation is required for intracellular trafficking of influenza B virus NB protein and efficient influenza B virus growth in vitro.

All influenza viruses bud and egress from lipid rafts within the apical plasma membrane of infected epithelial cells. As a result, all components of p...
902KB Sizes 0 Downloads 3 Views