(Received for publication, October 17, 1996)
From the Center for Gene Research and Biotechnology, Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804
Computer-assisted alignment of known
palmitylproteins was used to identify a potential peptide motif,
(TMD, transmembrane domain; X, any amino acid; C, cysteine
acceptor residues; A, aliphatic residue) responsible for
directing internal palmitylation of the vaccinia virus 37-kDa major
envelope antigen, p37. Site-directed mutagenesis was used to confirm
this motif as the site of modification and to produce a nonpalmitylated
version of the p37 protein. Comparative phenotypic analysis of the
wild-type and mutant p37 alleles confirmed that the p37 protein is
involved in viral envelopment and egress, and suggested that attachment of the palmitate moiety was essential for correct intracellular targeting and protein function.
Palmitylation involves the dynamic (reversible) post-translational addition of a 16-carbon saturated fatty acyl moiety via thioester or ester linkage to cysteine, serine, or threonine residues. Protein palmitylation has been demonstrated to play a variety of roles in vivo. When the function of palmitylation can be established, it usually falls into one of three categories. (i) Palmitylation can "activate" a protein, usually by targeting the protein to a site where it carries out its function (1) (the protein is then "inactivated" by depalmitylation); (ii) palmitylation can mediate protein-protein interactions (2); or, as is most often the case, (iii) palmitylation mediates protein-membrane interactions (2).
There are many examples of cellular and viral palmitylproteins (for reviews see Refs. 2-7). In many cases the palmitate acceptor residues have been identified, but a sequence or structural motif specifying palmitylation of proteins remains elusive. Recently a thioesterase demonstrated to cleave the palmityl group from proteins has been purified and cloned, but the enzyme(s) responsible for the addition of palmitate to proteins has not been identified. In some cases, palmitylation of one protein is dispensable for function, while on closely related proteins it is absolutely necessary for function. For example, the hemagglutinin of influenza virus is palmitylated on conserved cysteine residues of its cytoplasmic tail. For the H1 subtype of influenza, a partial block on palmitylation resulted in attenuation, and mutants in which the hemagglutinin was not palmitylated were not viable (8). Surprisingly, the hemagglutinin of the H3 subtype does not require palmitylation for the production of infectious particles (9). A similar disparity is found among cellular palmitylproteins. Palmitylation of the Src family of tyrosine kinases targets them to caveolae (1), while palmitylation of caveolin (a normally palmitylated constituent of caveolae) is not necessary for targeting to that structure (10).
Vaccinia virus (VV)1 is a member of the Poxviridae, a family of large, complex DNA viruses that replicate in the cytoplasm of infected cells (11). With the eradication of smallpox, the focus on VV biology momentarily faded, but it has been "rediscovered" as a versatile tool for molecular biologists. This has spurred recent efforts to characterize the virus. The 191-kilobase pair genome has been completely sequenced (12) and appears to encode nearly 200 gene products. Many are involved in nucleic acid metabolism, immune modulation, or serve some other non-structural function, and about 100 are structural proteins that are packaged with the virion. The infectious virion may exist in one of four forms; intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). IMV is the simplest in structure, being composed of the core particle and a double-layered membrane derived from the intermediate compartment between the endoplasmic reticulum and the Golgi stacks (13). By a process that is still poorly understood, IMV particles are targeted to the trans-Golgi and subsequently enwrapped by a double membrane derived from it (14, 15) resulting in the production of IEV. Virions then migrate to the cell surface, where by fusion and loss of the outermost membrane, followed by release into the medium, EEV are produced. If the virions remain attached to the outer face of the plasma membrane, they are referred to as CEV. This process is mediated by numerous VV-encoded proteins (16-20), including p37 and a 42-kDa glycoprotein (gp42).
Acylation of VV polypeptides has been demonstrated to play a major role in the assembly of virions. At least four proteins are myristoylated on glycine of the amino-terminal motif, methionine-glycine-(any 3 amino acids)-serine/threonine. The most studied of these is the 25-kDa protein product of the L1R open reading frame (21). Expression of a nonmyristoylated L1R protein within infected cells rather than the normally acylated protein resulted in a one-log reduction in titer relative to wild-type virus. It has also been previously demonstrated that there are at least six palmitylproteins induced in VV-infected cells (22). They are all present in the membrane fraction of cells and when virion-associated, are present in the outer membrane fraction of CEV and EEV. Three remain unidentified, and the others are encoded by VV open reading frames A34R (23), B5R (18), and F13L (14, 24), which express a 21-26-kDa glycoprotein, gp42, and the object of this study, p37, respectively.
Inactivation of the parental genes for either p37 or gp42 proteins in the VV genome results in varying degrees of inhibition on the envelopment and release of enveloped virions. One such deletion mutant is vRB10 (16), a derivative of the IHD-J strain of vaccinia. In this mutant, 93% of the F13L open reading frame (encoding p37) has been replaced with the gpt gene under the control of the VV 7.5-kDa promoter (p7.5K). During the characterization of this recombinant, a striking biological defect was noted. While producing normal amounts of IMV, the production of enveloped virus was severely inhibited. Additionally, the virus was not able to efficiently spread (in tissue culture) from cell to cell. Most strains of VV produce visible plaques within 24 h after infection of a permissive cell monolayer, but vRB10 requires at least 4 days before minute plaques are visible. Clearly, p37 is a major contributor to the envelopment and release process.
p37 is a 372-amino acid polypeptide expressed at late times during infection and has a predicted mass of 41 kDa. In gels it has an apparent mass of 37 kDa, which has been attributed to modifications that alter its electrophoretic mobility. Within infected cells, p37 localizes to the trans-Golgi network (TGN; Ref. 15) where it is tightly associated with the outer face of the organelle. The significance of the palmitate moiety in mediating membrane attachment has been demonstrated in vitro (25), and we sought to confirm and extend that work in vivo.
We are interested in several aspects of p37 palmitylation. The first is to define a motif that specifies palmitylation of VV proteins and perhaps refine it to include all viral and cellular palmitylproteins. Second, we wish to determine the biological function of p37 palmitylation as it pertains to activity of the protein, protein-protein interactions, or targeting to cellular or viral structures. We report here that we have defined a motif specifying palmitylation of p37. Furthermore, palmitylation of p37 is required for correct targeting to intracellular membranes, a prerequisite to function. Our results are discussed within the context of VV biology as well as the general aspects of eukaryotic protein acylation.
BSC40 (African green monkey kidney) cells were maintained in Eagle's minimal essential medium (MEM-E) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine (LG), 10 µg/ml gentamycin sulfate (GS) at 37 °C in a 5% CO2 humidified atmosphere. The IHD-J strain of VV was routinely propagated and titered in BSC40 cells as described previously (26). The mutant virus vRB10 (also referred to as I-VP37) has been described previously (16). It is a derivative of the IHD-J strain in which 93% of the gene encoding p37 has been replaced by the mycophenolic acid resistance gene gpt under the control of p7.5K. vRB10 was propagated by low multiplicity passages through BSC40 cells in the presence of mycophenolic acid, xanthine, and hypoxanthine. Virus stocks were titered by inoculating serial dilutions onto confluent monolayers of BSC40 cells subsequently transfected with plasmid DNA encoding a rescuing copy of the p37 gene (see below). Plaques were visualized by staining infected monolayers with crystal violet 72 h post-infection (HPI).
Computer Analysis of p37The Net Entrez program was used to find and retrieve the gene and amino acid sequence to p37 from VV strain IHD-J. The sequence was analyzed using the Genetics Computer Group suite of programs to generate a Hopp-Woods (27) hydrophilicity profile and a Chou-Fasman (28) secondary structure prediction. The sequence was then submitted for analysis by TMPred (29)2 to identify potential transmembrane-spanning regions of the protein.
Construction of p37-encoding Transient Expression VectorsPlasmid pDG3.0 is a pUC118-based vector that contains the
VV tk gene into which p7.5K has been inserted. Plasmids
pDG4.0, pDG4.1, pDG4.2, and pDG4.3 all have the VV F13L open reading
frame cloned adjacent to p7.5K in pDG3.0 such that transcription
initiating from p7.5K results in the production of an mRNA that
encodes p37. Plasmid pDG4.0 encodes the wild-type p37 protein.
Oligonucleotide-directed mutagenesis (30) was used to mutate the F13L
sequence in pDG4.0 to generate plasmids pDG4.1, pDG4.2, and pDG4.3. The
oligonucleotide oDG4.1 (5-GCTAACTGGCAGACAAGAAGCTGCAGAGC-3
) was used
to construct pDG4.1. It mutates the codon encoding cysteine 185 of p37
to encode serine and introduces a PstI restriction site by
silent mutation. Plasmid pDG4.2 was constructed using the
oligonucleotide oDG4.2 (5
-GCTAACTGGCAGAGAACAAGCTGCAGAGC-3
). It
mutates the codon encoding cysteine 186 of p37 to encode serine and
introduces a PstI restriction site by silent mutation.
Plasmid pDG4.3 was constructed using oligonucleotide oDG4.3
(5
-GCTCTGCGGCTTCTTCTCTGCCAGTATGC-3
). The oligonucleotide oDG4.3
mutates the codons encoding cysteines 185 and 186 to encode serine. The
mutations were confirmed by DNA sequencing.
Subconfluent monolayers of BSC40 cells in
35-mm wells were infected with either IHD-J or vRB10 at a multiplicity
of infection (m.o.i.) of 10. Concurrent with infection, vRB10-infected
cells were transfected with plasmids pDG3.0, pDG4.0, pDG4.1, pDG4.2, or
pDG4.3. Liposome-mediated transfection following the method of Rose
et al. (31) with modifications described by Campbell (32)
mediated the transfer of plasmid DNA and transient expression of p37.
The inoculum was prepared in polystyrene tubes to which was added 500 µl of MEM-E, the appropriate amount of virus, 30 µl of liposomes,
and 10 µg of the appropriate DNA. When no DNAs were to be
transfected, liposomes were added to the inoculum as a control. After
this mixture remained at room temperature for 15 min, it was added to
the cell monolayer from which the culture medium had been aspirated.
The cells were placed at 37 °C for 4 h, after which the
inoculum was aspirated and replaced with 1 ml of MEM-E with 3% FCS,
LG, and GS. At 6 HPI, the culture supernatant was aspirated and the
cells washed with warm MEM-E. The wash was aspirated and replaced with
1 ml of MEM-E containing 200 µCi of [9,10-3H]palmitic
acid ([3H]PA, DuPont NEN) or 1 ml of MEM-E containing 200 µCi of [9,10-3H]oleic acid ([3H]OA,
DuPont NEN). At 24 HPI the cells were harvested in the culture supernatant and transferred to microcentrifuge tubes. The samples were
centrifuged at 15,000 × g for 30 min to pellet cells
and any virions released during the infection. The pellet was
resuspended in 60 µl of phosphate-buffered saline (PBS) and
freeze-thawed three times. Each cell extract was divided into three
fractions of 20 µl each. The first fraction was prepared for
immunoprecipitation by addition of 500 µl of a 2 × strength
radioimmunoprecipitation assay buffer (1 × RIPA: 1% w/v sodium
deoxycholate, 1% v/v Triton X-100, 0.2% w/v SDS, 150 mM
sodium chloride, 50 mM Tris-HCl, pH 7.4, and 1 unit/ml
BenzonaseTM endonuclease) and incubation on ice for 15 min. The samples
were then heated to 70 °C for 2 min, followed by centrifugation at
6500 × g for 2 min. The supernatant was transferred to
new microcentrifuge tubes and the RIPA adjusted to 1 × concentration with water. Anti-p37 antiserum (-p37) was added to the
extracts, followed by incubation on ice for 2 h. 40 µl of a 50%
slurry of protein A-Sepharose beads in 1 × RIPA were then added,
and incubation was continued for 18 h at 4 °C with constant
agitation. The immunoprecipitated proteins were washed three times with
1 × RIPA, transferred to a new microcentrifuge tube, and washed
again. The beads were pelleted a final time and resuspended in reducing
sample buffer and boiled for 3 min. The proteins were resolved by
discontinuous gel electrophoresis (SDS-PAGE), utilizing 12%
polyacrylamide gels as described previously (33). Following
electrophoresis the gels were fluorographed by impregnation with 22.2%
PPO in Me2SO (34), drying, and exposure to Kodak BIOMAX MR
film at
70 °C. The second and third fractions from the total cell
extracts were directly subjected to SDS-PAGE as described above. One
gel was fluorographed as above to detect all vaccinia-encoded
[3H]PA- or [3H]OA-labeled proteins. The
other gel was subjected to immunoblot analysis (35) by blotting it to a
nitrocellulose filter, which was then probed with
-p37.
Antigen-antibody complexes on the filter were detected by incubation
with goat anti-rabbit antiserum conjugated to alkaline phosphatase
(Bio-Rad) and development with 5-bromo-4-chloro-3-indolyl phosphate and
p-nitro blue tetrazolium chloride.
BSC40 cells were grown to 95%
confluence in 100 mm tissue culture dishes. The cells were infected
with IHD-J or vRB10 at high multiplicity (m.o.i. = 10) or low
multiplicity (100-300 plaque-forming units/plate). Concurrent to
infection with vRB10, the cells were transfected with 300 µl of
liposomes and 100 µg of pDG3.0, pDG4.0, or pDG4.3 DNA. Low
multiplicity infection/transfections were done in duplicate. The cells
were placed at 37 °C for 4 h, after which the inoculum was
aspirated and the cells washed once with warm MEM-E. Then 10 ml of
MEM-E with 2.5% FCS, LG, and GS were added to each plate, and the
cells were placed at 37 °C for the remainder of the experiment. At
24 HPI the cells infected at a high m.o.i. were harvested and assayed
for p37 production by SDS-PAGE and immunoblot analysis using -p37
essentially as described above. Additionally, one set of the duplicate
plates infected at a low m.o.i. was stained with crystal violet (0.1%
crystal violet in 30% ethanol) at 24 HPI, while the infection of the
other set was allowed to continue until 72 HPI. They were then stained
with crystal violet as well.
BSC40 cells were
grown to 95% confluence in 100-mm tissue culture dishes. The cells
were infected with IHD-J or vRB10 at an m.o.i. of 10. Concurrent to
infection with vRB10, the cells were transfected with 300 µl of
liposomes and 100 µg of pDG3.0, pDG4.0, or pDG4.3 DNA. The cells were
placed at 37 °C for 4 h, after which the inoculum was aspirated
and the cells washed once with warm MEM-E. Then, 10 ml of MEM-E with
2.5% FCS, LG, and GS were added to each plate and the cells were
placed at 37 °C. In a duplicate experiment,
[35S]methionine/cysteine (DuPont NEN) was added to the
culture to a final concentration of 25 µCi/ml in the culture
supernatant to label nascent peptides. At 12 HPI the culture medium was
aspirated and the cells washed free of the plates with 5 ml of ice-cold PBS. The cells were pelleted by centrifugation at 700 × g for 10 min, followed by fractionation utilizing
differential centrifugation essentially as described by Child and Hruby
(22) with modifications as follows. The PBS was aspirated and the cells
resuspended in 2.0 ml of hypotonic buffer (HB: 20 mM HEPES,
pH 7.6, 5 mM potassium chloride, 1 mM magnesium
chloride, 150 mM sodium chloride) and incubated 10 min on
ice to swell the cells. All subsequent steps were at 4 °C. The cells
were then lysed by Dounce homogenization. One half of the cell lysate
(1.0 ml) was set aside as the total cell extract, while the remainder
was centrifuged at 700 × g for 10 min. The supernatant
(post-nuclear supernatant, PNS) was transferred to a new
microcentrifuge tube for further fractionation, while the pellet from
that centrifugation was resuspended in 1.0 ml of HB and set aside as
the nuclear pellet (NP). The PNS was centrifuged at 15,000 × g for 30 min. The pellet from this centrifugation was
resuspended in 1 ml of HB and set aside as the virus-containing fraction (P15). The supernatant from this centrifugation was
transferred to an ultracentrifuge tube and diluted to 4.5 ml with HB
and ultracentrifuged at 100,000 × g for 60 min. The
pellet from this centrifugation was resuspended in 1 ml of HB and set
aside as the subcellular organelle/cytosolic aggregate fraction (P100).
The supernatant from this centrifugation was adjusted to be 10%
trichloracetic acid and centrifuged at 15,000 × g for
30 min. The pellet from this centrifugation was set aside as the
soluble cytosolic fraction (S100). All other fractions were adjusted to
be 10% trichloracetic acid and centrifuged at 15,000 × g for 30 min. All precipitated pellets were resuspended in
100 µl of 1 M Tris, pH 10.0, to neutralize the acid and
briefly sonicated to facilitate resuspension. Twenty µl of each
fraction were analyzed by SDS-PAGE and immunoblot using -p37 (12%
polyacrylamide gel) as well as antiserum directed against the VV major
core protein precursor P4a (
-P4a, 10% polyacrylamide gel) and the
VV-encoded thymidine kinase (
-TK, 15% polyacrylamide gel).
Detection of the rabbit antibody-protein complexes (
-p37,
-P4a,
-TK) was by color development as described above or by chemiluminescence. For chemiluminescent detection, the primary antibody
incubation was followed by incubation with goat anti-rabbit IgG
horseradish peroxidase conjugate (Pierce). The protein-antibody complexes were detected by incubation with a chemiluminescent peroxidase substrate and exposure to film. Relative quantitation of
protein-antibody complexes was performed by film densitometry. To
determine the subcellular fractions that contained gp42, cells were
infected/transfected as outlined above. The nascent peptides were
labeled from 4 until 12 HPI by addition of
[35S]methionine/cysteine. At 12 HPI the cells were
harvested in PBS and fractionated as described above. Each fraction was
subject to immunoprecipitation using a mouse monoclonal antibody to
gp42 (
-gp42) essentially as described above. The immunoprecipitated proteins were resolved by SDS-PAGE and fluorographed. The
immunoprecipitates were quantitated by film densitometry.
BSC40 cells were
seeded onto microscope slide coverslips ("microcover glasses," VWR)
in 35-mm wells at a density of 5 × 105 cells/well and
cultured at 37 °C for 18 h. They were then infected with either
IHD-J or vRB10 at an m.o.i. of 10. The vRB10-infected cells were
concurrently transfected with either pDG3.0, pDG4.0, or pDG4.3 as
described above. At 12 HPI the culture medium was aspirated and the
cells washed once by incubation for 5 min in ice-cold PBS. The wash was
aspirated, and the cells were fixed to the microcover glasses by
incubation at 20 °C in 100% methanol for 20 min.
Immunofluorescent antibody labeling techniques (adapted from Ref. 36)
were employed to detect the presence and localization of VV proteins.
The cells were washed by two 5-min incubations in ice-cold PBS and then
incubated for 2 h at 4 °C in 1 ml of primary antibody
solution/well. The antibody solution was prepared as 1:1000 dilutions
of either
-p37,
-gp42 or a mixture of
-p37 +
-gp42 in PBS + 5% (v/v) FCS. The primary antibody solution was then aspirated, and
the cells washed four times with ice-cold PBS. Five hundred µl of
secondary antibody solution were added to each well, and the cells were
incubated for 2 h at 4 °C. The cells that had been incubated
with the rabbit-produced
-p37 antiserum were incubated with goat
anti-rabbit fluorescein isothiocyanate conjugate (G
R-FITC, 1:100
dilution in PBS + 5% FCS, Accurate Chemical & Scientific Corp.) as the
secondary antibody. The cells that had been incubated with mouse
monoclonal
-gp42 were incubated with goat anti-mouse
tetramethylrhodamine isothiocyanate conjugate (G
M-TRITC, 1:200
dilution in PBS + 5% FCS, Accurate Chemical & Scientific Corp.) as the
secondary antibody. The cells that had been incubated with the
-p37 +
-gp42 primary antibody mixture were incubated with a secondary
antibody mixture of G
R-FITC (1:100 dilution) + G
M-TRITC (1:200
dilution) in PBS + 5% FCS. Following the secondary antibody
incubation, the cells were washed four times for 5 min each with
ice-cold PBS. After the last wash, the microcover glasses were removed
from the tissue culture dishes and allowed to air-dry for 10 min before
mounting on microscope slides. Microscopic observations were with a
Zeiss photomicroscope through a 10× eyepiece and a 40× oil immersion
lens. The rabbit antibody-antigen complexes were visualized by
excitation of the FITC fluorophore at 492 nm and observation of
fluorescence through a 520-nm filter. The mouse antibody-antigen
complexes were visualized by excitation of the TRITC fluorophore at 550 nm and observation of fluorescence through a 570-nm filter.
Photographic images were captured on Kodak TMAX 100 black and white
film using a Zeiss M35 camera with exposure control by a Zeiss MC 63 on
semi-automatic control.
The in vitro analysis of p37 by Schmutz et al. (25) confirmed the contribution of acylation to the overall biochemical nature of the p37 protein. The protein is very hydrophobic and tightly associated with membranes. Detergent extraction of p37-containing membranes results in p37 partitioning to the detergent-enriched phase normally, but if the extracts are first treated with hydroxylamine, p37 partitions to the aqueous phase. To determine the function of the p37 palmitate moiety in vivo, we sought to construct a nonpalmitylating p37 mutant and express it within VV-infected cells and compare its behavior and activity to the wild-type protein.
Computer Analysis of p37Palmitylation involves the
post-translational addition of a 16-carbon saturated fatty acyl moiety
via thioester or ester linkage to cysteine, serine, or threonine. Child
and Hruby (22) have demonstrated by reverse-phase high performance
liquid chromatography that the modifying fatty acid is in fact
palmitate and that the palmitate-p37 bond is labile in the presence of
hydroxylamine, suggesting that modification occurs through thioester
linkage to a cysteine residue. The amino acid sequence of p37 was
deduced from the sequence of the VV strain IHD-J F13L gene published by Schmutz et al. (37). The protein contains 11 cysteine
residues, occurring at amino acid positions 13, 35, 53, 54, 98, 120, 129, 181, 185, 186, and 297 of the 372-amino acid polypeptide. The entire amino acid sequence of p37 was analyzed to predict regions of
hydrophobicity, secondary structure, and putative transmembrane regions. Overall, the protein is unremarkable in structure, as predicted by computer-assisted analysis. The central part of the protein contains two major hydrophobic domains, one of which is predicted to be a transmembrane domain as well (Fig. 1).
The protein is predicted to consist of 36% -helices, 30%
-strands/sheets, 21% turns, and the remaining 13% in other
structures.
A motif that specifies palmitylation of viral glycoproteins proteins has been previously defined by Ponamaskin and Schmidt (7), but putative identification of the modified cysteine residue(s) could not be made by sequence analysis of p37 alone. However, to facilitate identification of the palmitylation site of p37, we examined numerous palmitylproteins whose site(s) of modification is (are) known in order to potentially refine the reported motif specifying viral glycoprotein palmitylation. Taking into account our analyses of the sequences, secondary structures, and membrane topologies of other palmitylated proteins (data not shown) and previously reported structural requirements for palmitylation, we have arrived at a loosely conserved motif and based our prediction of the palmitylation site(s) of p37 on that. Palmitylation, it seems, occurs most often on cysteine residues 3 to 14 residues downstream (on the cytoplasmic side) of a transmembrane region. The cysteine residue(s) is (are) preceded by two aliphatic residues and is (are) followed by another aliphatic residue. In short, our motif is defined as TMDX1-12AAC(C)A, where TMD is transmembrane domain, X is any amino acid, A is any aliphatic amino acid, and C is the palmitylated cysteine(s). On the basis of these criteria, a single region of p37 is predicted to be palmitylated. The cysteine doublet occurring at positions 185 and 186 of p37 is within a predicted hydrophobic transmembrane region. The cysteines are preceded by two alanines and followed by a leucine. Although this does not strictly adhere to the structural requirements of the motif, it most closely resembles it. Additionally, the structural requirements defined by Ponamaskin and Schmidt (7) allow for palmitylation of cysteine residues within transmembrane domains but are limited to those occurring within six amino acids from the cytoplasmic border. Cysteines 185 and 186 are located 11 and 10 amino acids away, respectively, from the predicted cytoplasmic border. No other cysteine residues were possible candidates for palmitylation based on our predictions; therefore, we tested this region as the site of palmitylation by site-directed mutagenesis of the cysteine residues predicted to be palmitylated and transient expression of the resulting mutant proteins.
Identification of the Acylation Site of p37The recombinant virus, vRB10, a derivative of the IHD-J strain of VV, has had the F13L open reading frame insertionally inactivated and 93% deleted by an exogenous expression cassette mediating antibiotic resistance. This recombinant, although viable in tissue culture, does not produce p37 and consequently does not produce enveloped virions (CEV or EEV) or form plaques on susceptible monolayers of cells as efficiently as the wild-type virus. That these deficiencies were due to the loss of a functional p37 was proven by the restoration of the wild-type phenotype after reinsertion of the F13L open reading frame back into the genome of vRB10 (16). This virus is able to direct the transient expression of proteins provided that the gene encoding them is adjacent to a VV promoter. This is the basis of our analytical system.
We have constructed transient expression vectors that when transfected
into VV-infected cells are able to mediate the expression of either the
wild-type p37 or mutant forms of p37 containing cysteine to serine
permutations at positions predicted to be palmitylated (see Table
I). By infection with vRB10, transfection with
p37-encoding transient expression vectors, followed by addition of
[3H]PA to the cultures, we were able to observe
incorporation of label by VV proteins. After harvesting the total cell
extracts from infected cells, we first analyzed them for endogenous or transient expression of p37 (Fig. 2A) by
SDS-PAGE and immunoblot using -p37. We found that p37 was expressed
efficiently from all of our transient expression vectors and from
wild-type VV without any p37 being expressed from vRB10. We also
analyzed an equivalent fraction of the same extracts for incorporation
of labeled palmitate by total VV proteins using SDS-PAGE and
fluorography. The profile of palmitylated proteins expressed by vRB10
is identical to IHD-J with p37 being the obvious exception (Fig.
2C), although another palmitylprotein of approximately 37 kDa is present. This protein incorporates palmitate less efficiently
than p37, but to confirm that it was not p37 from a contaminating
source, we then immunoprecipitated labeled p37 from the same total cell
extracts and analyzed the immunoprecipitate by SDS-PAGE and
fluorography (Fig. 2B). No labeled proteins were detected in
the immunoprecipitate from vRB10-infected cells. A single protein (p37)
was detected in the IHD-J-infected cell extracts as well as the cell
extracts in which transient expression of wild-type p37 was mediated by vRB10. Labeled p37 was also detected in vRB10-infected cell extracts where transient expression of p37 was from plasmids pDG4.1 and pDG4.2.
The proteins expressed from these plasmids contain mutations that
exchange individual cysteines at positions 185 or 186 for serine.
Labeled p37 could not be detected in cells that were transfected with
pDG4.3, suggesting that the protein expressed from this plasmid is not
palmitylated. The protein encoded by pDG4.3 is a mutant that has both
cysteines at positions 185 and 186 of p37 replaced by serine. We have
concluded that cysteines at position 185 and 186 of p37 are both
modified by palmitate and that they are the only sites on the protein
that are modified.
|
Nonpalmitylated p37 Does Not Rescue Plaque Formation by vRB10
Cell-to-cell spread of VV is primarily by EEV and CEV with
little contribution from IMV. As a consequence of inactivating the F13L
open reading frame, the recombinant virus vRB10 was rendered inefficient at forming plaques on monolayers of cells (16). The process
of envelopment and release of virions were efficiently rescued by
transient expression or marker transfer of DNA encoding a functional
p37. Thus, the ability to rescue plaque formation by vRB10 is an
excellent measure of the production of a functional p37 within infected
cells. In our experiments, we infected cells with the wild-type IHD-J
or vRB10 at high or low m.o.i. Both the high and low m.o.i. infections
were concurrently transfected with DNA encoding the wild-type p37
(pDG4.0) or a nonpalmitylating p37 mutant (pDG4.3) under identical
conditions. At 24 HPI the cells infected at high m.o.i. were assayed
for p37 production by immunoblot analysis using -p37 as the primary
antibody. The protein was expressed in the wild-type IHD-J infected
cells and in cells transfected with pDG4.0 or pDG4.3 (data not shown).
No p37 was detected in the vRB10-infected cells. Also at 24 HPI, one
set of cells infected at low m.o.i. was stained with crystal violet to
detect plaques (Fig. 3). IHD-J formed visible plaques at
24 HPI and was just beginning to form the characteristic comet tail-shaped plaques due to the release of EEV. At 24 HPI, no plaques were visible, except by light microscopy, in cell monolayers infected with vRB10 regardless of which DNA was transfected (data not shown). At
72 HPI the second set of identically infected/transfected cells was
stained with crystal violet. The IHD-J-infected cell monolayer was
completely obliterated, while only very minute plaques were visible in
the vRB10-infected cell monolayer. The monolayer of vRB10-infected
cells that were transfected with the wild-type p37-expressing vector
had plaques equal in size to IHD-J-infected cell monolayers at 24 HPI,
with large comet-shaped plaques occurring at an approximate frequency
of 1:100 relative to the smaller plaques. The vRB10-infected cell
monolayers that were transfected with the vector encoding the
nonpalmitylated mutant p37 had only very minute plaques equal in size
to those plaques formed in vRB10-infected cell monolayers. No large
comet-shaped plaques were observed. By this assay, it seems that a
nonpalmitylated p37 is non-functional with regard to envelopment and
release of infectious virus, events that are necessary for plaque
formation.
The Palmitate Moiety on p37 Mediates Its Membrane Interaction
It has been previously demonstrated that within infected cells, p37 is associated with TGN membranes (15) and that when virion-associated, is found exclusively on the inner face of the EEV outer envelope (25, 38). In vitro analysis of the function of the palmitate moiety on p37 suggests that it is the fatty acid that mediates membrane affinity and not the computer-predicted hydrophobic domains in the protein. Schmutz et al. (25) performed detergent partitioning studies using normal palmitylated p37 and p37 that had been treated with hydroxylamine to hydrolyze the labile thioester linkage between p37 and the palmitate moiety. The protein was found to partition to the aqueous phase when not palmitylated but was normally found in the detergent phase.
We sought to confirm the in vitro findings by an in
vivo analysis. First we performed differential centrifugation
subcellular fractionation of infected cells. Cells were infected with
IHD-J or vRB10 at a high m.o.i. The wild-type p37-encoding plasmid or a
nonpalmitylating p37 mutant-encoding plasmid were transfected into
vRB10-infected cells. At 12 HPI the cells were fractionated as outlined
under "Experimental Procedures" to yield a nuclear fraction (NP)
and a cytoplasmic fraction (PNS), which was further fractionated into a
virus-containing fraction that potentially contains some of the
cytoplasmic membrane-bound organelles (P15), a particulate cytoplasmic
fraction that has been depleted of virus by the P15 (P100), and a
soluble cytoplasmic fraction (S100). We assayed all the fractions for
the presence of p37 as well as gp42, the VV major core protein 4a, and
the VV-encoded thymidine kinase (TK) as controls. There was no
discernible difference between IHD-J-infected cells and vRB10-infected
cells as far as the fractionation of 4a and TK were concerned (data not
shown). Specifically, the precursor to 4a (P4a) and 4a were found at
approximately the same concentration in the NP as in the PNS. When the
PNS was fractionated further, P15 favored 4a over P4a while little of
either P4a or 4a was found in the P100. Surprisingly, a significant
amount of P4a was found in the soluble cytosolic fraction. Thymidine
kinase was found to be present in the PNS and the S100 fractions as
would be expected of a soluble enzyme and demonstrates that complete cell lysis has occurred. Within IHD-J-infected cells (Fig.
4A), 87% of p37 was present in the NP,
11.5% in the P15, and less than 2% total in the P100 and the S100
fractions. In cells in which the wild-type p37 was transiently
expressed (Fig. 4C), 68% of p37 was present in the NP, 30%
in the P15, and 1% in both the P100 and S100 fractions. The
nonpalmitylated p37 (Fig. 4D) was present in all fractions.
The NP contained 48%, the P15 contained 15.5%, the P100 contained
2%, and the S100 contained 34.5%. Approximately half of the gp42
present in cells was found in the NP regardless of the palmitylation
state of p37. When p37 was palmitylated (Fig. 4, E and
G), an approximately equal portion was present in the viral
pellet (P15), but when p37 was not present or not palmitylated (Fig. 4,
F and H), gp42 was equally distributed among the
P15, P100, and S100 fractions.
The second method to determine the function of the palmitate moiety was
by immunofluorescent analysis of infected cells. The localization of
p37 within infected cells has been previously demonstrated by
immunofluorescence (14). When cells have been incubated with
fluorescent antibodies directed against p37, a punctate cytosolic
pattern of fluorescence is observed, which is indicative of association
with cytosolic membrane-bound compartments. We have demonstrated that
p37 co-localizes with gp42 (Figs. 4 and 5), another
VV-encoded protein that is necessary for envelopment and release of
virions. We have observed previously that when this co-localization is
disrupted by brefeldin A, no envelopment or egress of virus occurs
(39). We therefore sought to determine the intracellular localization
of both p37 (wild-type or nonpalmitylated) and gp42 within the same
cell as an analysis of correct localization for p37. Cells were
infected with IHD-J or vRB10 at a high m.o.i. The vRB10-infected cells
mediated the transient expression of wild-type or nonpalmitylated p37
from transfected DNA. The production and localization of p37 and gp42
were demonstrated by indirect immunofluorescence (Fig. 5). The antibody
directed against p37 is from polyclonal serum produced in rabbits,
while the antibody directed against gp42 is a mouse monoclonal
antibody. This allowed us to direct different fluorescent secondary
antibodies against the primary antibodies and thus detect the presence
of both antigens within the same cell. Within cells infected by IHD-J
or vRB10 that were transiently expressing wild-type p37, both p37 and
gp42 were present at distinct foci within the cytoplasm of the cell displaying a pattern of fluorescence that was identical for both proteins. At late times after infection (18 and 24 HPI; data not shown), p37 and gp42 were present at the periphery of the cell in
addition to cytoplasmic foci. Cells that were expressing the nonpalmitylated p37 displayed a diffuse cytosolic pattern of
fluorescence for p37 but gp42-specific fluorescence remained punctate.
We have concluded from this observation, as well as from the
fractionation studies, that palmitylation not only specifies membrane
affinity but that within infected cells targeting to appropriate
membranes is abrogated when the palmitate moiety is absent, resulting
in solubility in the cytoplasm.
In this study, we sought to examine the significance of
palmitylation of p37, a major protein constituent of VV EEV envelopes. To facilitate our study, transient expression vectors were constructed in which individual or multiple cysteine residues of p37 were replaced
by serine residues. The specific cysteine residues targeted for
permutation by serine were predicted to be the palmitate acceptor residues by contextual similarity to the putative palmitylation motif,
TMDX1-12AAC(C)A as
described above. The expression of these p37 mutants was mediated by a
p37 mutant VV (vRB10), which allowed analysis of
palmitate incorporation and functionality of the protein. Functional
analysis included rescue of the non-plaque-forming phenotype of vRB10
as well as experiments to determine the localization of the
nonpalmitylating p37 relative to wild-type p37.
Not only is p37 the most abundant protein constituent of the EEV envelope, but it appears to quantitatively incorporate more palmitic acid than other palmitylproteins of VV. This suggested to us that the protein may be modified at more than one site. By analysis of numerous palmitylproteins, both viral and cellular, we have defined a motif that we used to predict the palmitylation site of p37. Only one region of the protein was predicted to be palmitylated: a cysteine doublet occurring at positions 185 and 186 of the 372-amino acid polypeptide. Our motif is defined as TMDX1-12AAC(C)A with the palmitylated cysteine(s) (C) preceded by two aliphatic amino acids (A) and followed by another. This occurs most often on residues within or proximal to membrane-spanning domains (TMD). Our motif, as it turns out, is a refinement of one observed for viral glycoproteins (6, 7). Yang et al. (40) have observed that the human immunodeficiency virus and simian immunodeficiency virus gp41 protein, a glycosylated, sulfated, transmembrane protein that along with gp120 facilitates CD4 binding, is palmitylated, not proximal to its transmembrane-spanning region, but on cysteines that immediately precede and follow an amphipathic region known to associate with the cytoplasmic face of the plasma membrane. They postulate that palmitylation stabilizes the interaction of the amphipathic region with the membrane. Palmitylation of p37 may serve an analogous function.
Computer analysis predicts two hydrophobic domains in the protein, one possibly being a membrane-spanning region (Fig. 1). Considering the data presented in this study and the work of Schmutz et al. (25), it seems unlikely that p37 spans the membrane of the TGN or virion envelopes, even though it is tightly associated with them. Releasing p37 from the membrane requires only treatment with hydroxylamine, which cleaves the thioester palmitate-cysteine linkage. Additionally, the nonpalmitylating mutant described here is found to fractionate with the soluble cytoplasmic components of infected cells, as well as displaying a diffuse cytosolic pattern when examined by immunofluorescent microscopy. Our in vivo work confirms the in vitro analysis of p37. We have concluded that the membrane affinity of p37 is in large part mediated by palmitylation of cysteine residues at positions 185 and 186 of the polypeptide chain.
Our motif, as it seems, is imperfect and will require refinement in order to accurately predict the palmitylation sites of non-transmembrane-spanning proteins. Nevertheless, we were able to predict the palmitylation site of p37 based on the motif. When either cysteine 185 or cysteine 186 was replaced by serine, we observed a reduction in the efficiency of [3H]PA incorporation by p37 (Fig. 2B). The immunoblots of the same infected cell extracts (Fig. 2A) indicate that similar amount of protein were expressed relative to the transiently expressed wild-type protein, but the absolute efficiency of palmitylation at either cysteine residue was not measured.
In addition to modification by palmitate, p37 is subject to oleation (41). When VV-infected cells were cultured in the presence of [3H]OA and total cell extracts were analyzed by SDS-PAGE and fluorography, we observed incorporation of label by proteins with apparent masses of 14, 17, 23-28, 37 (p37 and an additional co-migrating 37-kDa oleated protein), 41, 56, 86, and 92 kDa (Fig. 2G). All of these proteins appear to co-migrate with known palmitylproteins of VV, suggesting that VV palmitylproteins belong to the class of "S-acylated" proteins that are preferentially palmitylated but alternatively are subject to modification by other long-chain fatty acids (reviewed in Refs. 6 and 42). To be certain that oleation of p37 did not occur at a site different than the palmitate acceptor site, we transiently expressed the nonpalmitylating p37 mutant in vRB10-infected cells in the presence of [3H]OA and assayed for incorporation of label by p37. Although the protein could be detected by immunoblot (Fig. 2E), it could not be detected by fluorography (Fig. 2F), suggesting that the protein was not modified by oleate if the palmitate acceptor cysteines were substituted with serine as would be expected for a S-acylated protein.
Palmitylation of p37 Is Required for Correct Localization and FunctionEnveloped virus, specifically CEV and EEV, are
responsible for cell-to-cell spread of VV and consequently plaque
formation on susceptible monolayers of cells. The requirement of p37 in this process has been previously established (43). When we transiently expressed wild-type p37 in vRB10-infected cells, plaques were formed,
albeit at a slower rate than for IHD-J. We initially attributed this to
one of two possibilities: (i) plasmid-mediated expression did not
provide the correct context for function, or (ii) expression kinetics
from p7.5K were sufficiently different from the F13L endogenous
promoter that normally synchronous events were perturbed. Examination
of infected cells by immunofluorescent microscopy suggested a third
explanation. Transfection efficiency, as determined by the ratio of
cells fluorescently labeled by -p37/G
R-FITC, ranged from 10 to
50% depending on which preparation of liposomes was used to mediate
the transfection. It stands to reason that cell-to-cell spread would be
inefficient under those conditions. On the other hand, cell-to-cell
spread by vRB10, when the only rescuing copy of p37 was not
palmitylated, was even less efficient. Throughout the course of the
experiment, the efficiency of plaque formation never exceeded that
observed for vRB10 when p37 is not palmitylated. One other phenotype
that we observed when the rescuing copy of p37 was wild-type, was the
formation of large comet-shaped plaques occurring at an approximate
frequency of 1:100 relative to the smaller plaques. We assume that this
is due to recombination between the plasmid copy of the F13L gene and
the VV genome, which contains intact 5
and 3
sequences of F13L.
Cell-to-cell spread would be much more efficient if the virus expressed
p37 from its genome instead of relying on the plasmid copy of the gene,
which is present in only 10-50% of the cells. When the
nonpalmitylating p37 is expressed in vRB10-infected cells, all plaques
formed were uniform in size. Considering that recombination frequency
appears to be about 1 in 100, there does not seem to be any selective advantage for recombinants that are expressing the nonpalmitylating p37
from their genomes. Further analysis of the significance of p37
palmitylation may require the isolation of recombinants that express
the nonpalmitylating p37 mutant. This may prove to be difficult
considering the lack of selection for recombinants and the pressure to
mutate back to wild-type once recombined.
We have considered the possibility that enveloped virions are formed and released but are not infectious. We have taken two approaches to address this possibility. First we attempted to purify virions from IHD-J- and vRB10-infected cells transfected with either wild-type or nonpalmitylated p37-encoding plasmids. We were able to demonstrate that IHD-J produced both IMV and EEV by CsCl gradients (data not shown) since they are separable by characteristic densities. While vRB10-infected cells produced normal amounts of IMV, the production of EEV could not be demonstrated even when a rescuing wild-type copy of p37 was expressed in those cells. We then attempted to assay for EEV-specific proteins in the infected cell culture supernatants. This proved to be even less sensitive, as we were unable to demonstrate conclusively, the presence of gp42 (data not shown) in IHD-J-infected cell culture supernatants. To address the possibility of noninfectious EEV release will probably require the construction of recombinant VV that expresses the nonpalmitylated p37, and, as alluded to above, this may prove difficult.
As to why the nonpalmitylating p37 mutant is not functional, we considered three possibilities. (i) Palmitylation of proteins has been demonstrated to activate a protein, usually by targeting the protein to a site where it serves its function (1). The protein is then inactivated by depalmitylation, resulting in diffusion away from the site of function. (ii) Palmitylation can mediate protein-protein interactions (2). (iii) Alternatively, as is most often the case, palmitylation mediates protein-membrane interaction (2). We do not consider palmitylation of p37 to be an activation switch. All of the p37 within a cell appears to localize to its site of function, specifically, intracellular membranes (Figs. 4, A and C, and 5), and by this study we know that interaction to be dependent on palmitylation of p37. The lack of any detectable (wild-type) p37 in the soluble cytoplasmic fraction of cells argues against regulation of p37 by reversible palmitylation.
There are reports that claim p37 covalently interacts with gp42 (41) and possibly noncovalently with the viral hemagglutinin (44). Recent work by Schmutz et al. (25) argues against either interaction. Within infected cells, p37 exists solely as a monomer, and in purified virions it exists as a monomer and a homodimer. The latter represents the minority of the total p37 present in EEV membranes, and its significance is not known. Considering that homodimerization occurs after envelopment and that the nonpalmitylating p37 mutant cannot mediate the envelopment and release of virions, it seems unlikely that the loss of function is due to the inability of p37 to dimerize.
This leaves membrane targeting/anchoring as the remaining possible function for palmitylation of p37. The work by Schmutz et al. (25) suggested as much in vitro, and our in vivo analysis confirms it. When VV-infected cells are fractionated by differential centrifugation, we find that wild-type p37 is concentrated in the particulate membrane-containing fractions (Fig. 4, A and C) in agreement with previously published reports (22). When p37 is not palmitylated, it is found in the membrane-containing fractions as well as the soluble cytosolic fractions (Fig. 4D). If the subcellular fractionation results are analyzed independent of the rest of this study, one might conclude that the palmitate moiety only serves to anchor p37 in a membrane that it has an intrinsic affinity for. This is a reasonable assumption but one that is not supported by immunofluorescent analysis of infected cells. Prior to virion association, p37 localizes to the TGN and by immunofluorescent analysis appears to co-localize with gp42 (Fig. 5) in agreement with immunoelectron microscopy work performed by Schmelz et al. (15). Indirectly labeling p37 or gp42 via fluorescently tagged antibodies results in identical punctate cytosolic foci of fluorescence within infected cells. If palmitylation of p37 served only to anchor the protein to its target membrane, one would expect that the nonpalmitylating p37 mutant would continue to co-localize with gp42 with little diffusion away from those sites. In fact, the opposite is true. The nonpalmitylating p37 mutant exhibits a diffuse cytosolic pattern of fluorescence with no discrete foci anywhere within infected cells. The pattern of fluorescence for gp42 remains undisturbed in cells expressing the nonpalmitylating p37 mutant, and, as such, p37 must not be involved in gp42 targeting to membranes. It is possible, though, that gp42 is involved in p37 targeting through interaction with its palmitate moiety as has been described for other protein-protein interactions (2).
Why though does most of the wild-type p37, and a significant portion of the nonpalmitylated p37, fractionate with the particulate fractions of the cell when it is thought to only associate with the TGN when palmitylated and remain soluble when palmitylation is blocked? Perhaps the lysis conditions favor aggregation in macromolecular structures, a possibility we have not investigated. It is also conceivable that the VV-induced reorganization of cytoskeletal structure (45) may alter the fractionation of subcellular components relative to uninfected cells. Nevertheless, a significant portion of the nonpalmitylated p37 found in the cytoplasmic fraction of infected cells is soluble and, when observed by immunofluorescence, displays a fluorescent pattern one would expect from a soluble cytoplasmic protein.
Vaccinia virus continues to stand out as a uniquely capable model system for the analysis of eukaryotic protein processing. In addition to acylation, VV polypeptides are subject to proteolytic processing, glycosylation, phosphorylation, ADP-ribosylation, disulfide cross-linking (reviewed in Ref. 46), and sulfation (41). We have yet to decipher all the intricacies of these modifications in VV or eukaryotic systems, and palmitylation of proteins is one of the least understood of these processes. Perhaps exploitation of the VV system will allow us to not only accurately predict protein palmitylation but identify factors mediating the process, including those responsible for the modification reaction and molecular properties of the modified protein.