Fitzpatrick et al. have suggested .... gC homologs is derived from Fitzpatrick et al. (5). ..... Spike- nucleocapsid interaction in Semliki Forest virus reconstructed.
Vol. 64, No. 7
JUlY 1990, p. 3516-3521 0022-538X/90/073516-06$02.00/0 Copyright X) 1990, American Society for Microbiology
JOURNAL OF VIROLOGY,
The Putative Cytoplasmic Domain of the Pseudorabies Virus Envelope Protein glll, the Herpes Simplex Virus Type 1 Glycoprotein C Homolog, Is Not Required for Normal Export and Localization KIMBERLY A. SOLOMON, ALAN K. ROBBINS, MARY E. WHEALY, AND L. W. ENQUIST* E. I. du Pont de Nemours & Co., Inc., Central Research and Development, Experimental Station, P.O. Box 80328, Wilmington, Delaware 19880-0328 Received 22 January 1990/Accepted 9 April 1990
Glycoprotein gIIl of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIll is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.
The glll glycoprotein gene of pseudorabies virus (PRV) is a member of a conserved gC glycoprotein gene family in herpesviruses initially described in herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) (23). This family of genes codes for structural glycoproteins found in the virus envelope and on the surface of infected cells. Seven members of this gene family have been sequenced, and their structural motifs have been analyzed by computer analysis (5). The genes are typical integral membrane proteins in that they have two hydrophobic domains, one at their amino terminus predicted to be a signal sequence and another at their carboxy terminus thought to be a transmembrane domain. The proteins are thought to span the membrane once, and all have short hydrophilic segments at their carboxy terminus predicted to be the cytoplasmic domain. Fitzpatrick et al. have suggested that members of the gC gene family are members of the immunoglobulin superfamily (5). To date, there is little evidence that any of the gC homologs have functional similarity, although attempts have been made to demonstrate complementation between HSV-1 gC and PRV
gIII (25). In general, predictions for functional domains within glycoproteins have been made by computer analysis and by comparison to known membrane proteins. Direct evidence concerning the functions of signal sequence, transmembrane, and cytoplasmic domains has only recently been accumulating for herpesvirus glycoproteins. Our laboratory has demonstrated that the amino-terminal hydrophobic domain of PRV gIII indeed functions as a signal sequence (4, 16). We have also provided evidence that sequences near the carboxy terminus of glll have a membrane-anchoring function (15). Holland et al. have demonstrated for HSV-1 gC that the membrane-anchoring domain is located near the carboxy terminus (6). The subject of this report concerns the function of the putative cytoplasmic domain of PRV gIII and its relationship to the other members of the gC gene family. Little is known about this domain for any gC homolog except HSV-1 gC. Holland et al. provided evidence that the cytoplasmic domain of HSV-1 gC was required for membrane anchoring (6). * Corresponding author. 3516
A deletion of the 3' end of the gene which removed 14 amino acids, including the predicted 11-residue cytoplasmic domain, resulted in secretion of the truncated protein into the media of infected cells. Removal of the putative cytoplasmic domain, however, did not affect the ability of the truncated protein to be localized to the virus envelope. In this study we sought to determine whether the predicted cytoplasmic domain of PRV gIII protein is similar to that of HSV-1 gC with respect to membrane-anchoring function and to discern whether the cytoplasmic domain functions in cellular localization and virion assembly of the gIII protein. Comparison of the putative cytoplasmic domains of the gC gene family members. Figure 1A depicts the glll protein and highlights the signal sequence (1 to 22) and the carboxyterminal hydrophobic (436 to 470) and cytoplasmic (471 to 479) domains. Figure 1B shows the carboxy-terminal residue alignment of the seven known gC homologs presented by Fitzpatrick et al. (5). Several points are important to consider. First, even though there is homology throughout the gC homologs, there is no striking consensus alignment after the hydrophobic domain, defined as a transmembrane sequence, except that a short stretch of hydrophilic amino acids follows the conserved hydrophobic sequences. Second, these short carboxy-terminal segments are called cytoplasmic domains largely due to prediction and not to direct experimental evidence. Third, the cytoplasmic domains have been defined by using different criteria in various laboratories. They can be defined as those sequences following the first positively charged amino acid after the hydrophobic stretch of amino acids predicted to be the transmembrane domain, or they can be defined as the inflection point on the hydrophilicity plot where the sequence switches from hydrophobic to hydrophilic. Fourth, in the optimum alignment of the homologs, a cysteine is conserved in five of the seven gC homologs very close to the beginning of the cytoplasmic domain, as defined by either method. It is interesting that the only two gC homologs lacking this conserved cysteine are HSV-1 gC and HSV-2 gC. Finally, the cytoplasmic domains in this alignment fall into two general categories with respect to positively charged residues. PRV glll and VZV gpV have only a single positively
NOTES
VOL. 64, 1990 A
3517
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.
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AGVLVV-TAIVYVVRTSQSRQRHRR* ..VAVVLAGTAVVYLTHASSVRYRRLR* .
*-
*
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FIG. 1. (A) Domains of the PRV glll glycoprotein. The regions of glll containing an uninterrupted stretch of hydrophobic amino acids are depicted as a black box. The putative cytoplasmic domain is shown as a hatched box. The length of these regions as well as the length of the entire gIII protein (in number of amino acids) is indicated above the diagram. The amino acid sequence of the C-terminal hydrophobic domain (436 to 470) and putative cytoplasmic domain (471 to 479) is shown below the map. (B) The alignment of the seven gC homologs is derived from Fitzpatrick et al. (5). Only the carboxy ends of the proteins, including the predicted cytoplasmic domains and part of the transmembrane domains, are shown. The conserved cysteines (C471 of PRV gIII) are boxed. Amino acids identical in four or more proteins are indicated by a dot below the sequence. The positively charged residues are in bold type.
charged residue, while the remaining five have three or more. Specifically, HSV-1 gC has five positively charged residues and BHV-1 glll has six. By using this alignment, we defined the conserved cysteine at residue 471 of PRV glll as a key feature defining the transition between the hydrophobic transmembrane region and the cytoplasmic domain. We therefore created two mutations at this site by using site-directed mutagenesis as described below. Generation of a unique EcoRI site in the gll gene near the cytoplasmic domain. Since all known strains of PRV, including the Becker strain used in this report (PRVBe), do not contain any EcoRI sites in their entire 150-kilobase-pair genomes (2, 15), we introduced an EcoRI site near the gIII cytoplasmic domain. In doing so, mutations could be introduced close to the EcoRI site and crossed back into the virus. Viruses carrying the unique EcoRI site would be likely to contain the site-directed mutation as well, thereby providing a means to screen for desired recombinant viruses. The wild-type PRV gIll gene (1,437 nucleotides) contains a unique BamHI site located between nucleotides 1376 and 1381 or 29 base pairs upstream from the coding sequence for the proposed cytoplasmic tail of the gIII protein (Fig. 2A) (12). It was possible to change the BamHI site (GGATCC) to a unique EcoRI site (GAATTC) at the same position without altering the codons. An EcoRI restriction site was created by changing the third G of the glycine codon GGG to an A and the C of the isoleucine codon ATC to a T. The method of Kunkel (8) was followed by using the Bio-Rad Muta-gene in vitro mutagenesis kit (Bio-Rad Laboratories), except that uracil-containing single-stranded DNA was obtained by transforming the dut ung double-mutant strain PR382 (dut-J ung-J thi relAl rpsE recA56 srl::TnJO F' lacl lacZ::TnS lacY lacA, constructed by J. Patrick Ryan) with the doublestranded plasmid pIG1. pIG1 is a derivative of pALM3 (13)
PRV1003
FIG. 2. Analysis of PRV recombinants. (A) PstI fragments containing the coding regions for glll from PRV-Be, PRV1001, PRV1002, and PRV1003. Relevent restriction endonuclease sites are indicated. The glll coding sequences are indicated by open boxes. The codon present at position 471 within each of the viral glll genes and the encoded amino acid are indicated by a triangle. The direction of transcription is indicated by the arrow. The distances are not sized to scale. (B) Southern blot analysis of nucleocapsid DNA (1) extracted from PRV-Be (Be), PRV1001 (1001), PRV1002 (1002), and PRV1003 (1003). The glll-specific fragments were analyzed by the Southern blot method (21) by using a glll-specific probe labeled with [a-32P]deoxyribonucleotide triphosphates by nick translation. The DNAs were digested with either PstI alone (lane 1) or PstI and EcoRI (lane 2). The sizes of the fragments were estimated by using DNA size standards and are indicated (in kilobase pairs) to the right of the blot.
containing the wild-type gIll gene plus 1 kilobase pair of downstream flanking PRV sequence. In addition, this plasmid contains the M13 intergenic region, which allows for the production of single-stranded DNA directly from the pIG1 plasmid without requiring subcloning into the M13 bacteriophage. This single-stranded DNA was used as a template to convert the BamHI site located within glll between nucleotides 1376 and 1381 to a unique EcoRI site by using the the primer containing mutagenic 22-base-pair sequence 5'GATGGCCAGAATTCCGATGCCG3' (see Fig. 2A). The mutagenized DNA was transformed into Escherichia coli NF1829 (12), and a plasmid containing an EcoRI site at the appropriate position was isolated and designated pKS1001. We show later that the creation of the EcoRI site in this fashion had no effect on the resultant gIII protein, as expected. The plasmid pKS1001, containing the gIII gene with the unique EcoRI restriction site, was used as the starting plasmid for site-directed mutagenesis of the gIII cytoplasmic tail. Isolation of recombinant viruses carrying mutations in the gIll cytoplasmic domain. To ascertain the role of the putative cytoplasmic domain of gIII in membrane anchoring, cellular localization, and virion assembly, two mutations were generated within this region of gIll by site-directed mutagenesis. Single-stranded DNA was prepared from plasmid pKS1001 and was used as a template to change the cysteine codon (TGC) at position 471 to either a nonsense amber codon (TAG) or a serine codon (AGC). The resultant plasmids were designated pKS1002 (cysteine to amber) and pKS1003 (cys-
3518
J. VIROL.
NOTES
teine to serine) (Fig. 2A). These mutations were produced as follows: pKS1001 was transformed into PR382, and uracilcontaining single-stranded DNA was isolated and used for another round of mutagenesis. The mutagenic primers used to generate pKS1002 and pKS1003 contained the sequences 5'GTAGTAGACCTACGTCGCCA3' and 5'GTAGTAGA CGCTCGTCGCCA3', respectively. Following transformation and segregation, the resultant mutagenized plasmid DNAs were sequenced from the poly(A) site of gIll to the XhoI site (nucleotide 1258) to confirm the presence of the mutations and to ensure that no other mutations were generated within this portion of gIll as a result of mutagenesis. To eliminate any second-site mutations that may have been generated in gIll upstream of the XhoI site, the plasmids containing the cytoplasmic domain mutations were digested with XhoI and PstI and the 4.3-kilobase fragments containing the C-terminal end of gIll were isolated from a 1% agarose gel and ligated with the 2.5-kilobase XhoI-Pstl fragments obtained from the parent pIGl plasmid. All restriction digests, DNA ligations, agarose gel electrophoresis procedures, and gel purifications of DNA fragments were done essentially as described by Maniatis et al. (10). The nucleotide sequence of the mutated regions was determined by the dideoxynucleotide chain termination method of Sanger et al. (17) by using the Sequenase kit (United States Biochemical Corp.). Restriction enzyme and DNA modifying enzymes were purchased from Bethesda Research Laboratories, Inc., and New England BioLabs, Inc. To analyze the phenotypes of the glycoproteins produced by the mutant gIII genes, the cytoplasmic tail mutations were crossed onto the PRV genome by homologous recombination. PRV2 genomic DNA, which contains a deletion of the 402-base-pair SacI fragment from the middle of gIll (13), was cotransfected into PK15 cells with either pKS1001, pKS1002, or pKS1003 DNA that had been digested with Ncol. Recombinants were detected with the black-plaque assay (7, 20) by virtue of their reactivity with the anti-gIII monoclonal antibody Ml (13, 16). Nucleocapsid DNA was isolated from a black-plaque-positive recombinant from each of the three transfections as well as from PRVBe and subjected to Southern blot analysis (21), as shown in Fig. 2B. These data show that the EcoRI site was indeed present in all three recombinants. Viral stocks of each mutant were made in PK15 cells and were designated PRV1001 (BamHI to EcoRI), PRV1002 (cysteine to amber), and PRV1003 (cysteine to serine). Export kinetics of gIll proteins expressed from PRV1001, PRV1002, and PRV1003 recombinant viruses. The kinetics of processing and export of the gIII glycoprotein produced from PRV1001, PRV1002, and PRV1003 were measured by pulse-chase analysis. PK15 cells, infected with either PRVBe or PRV1001 (multiplicity of 10), were labeled for 2 min with [35S]cysteine and then chased with growth medium containing excess nonradioactive cysteine for various periods of time (15). The extracellular medium cleared of virus and infected cell fractions were collected, and the gIIIspecific proteins were immunoprecipitated from each fraction by using the gIII-specific antiserum 282 (15). Following immunoprecipitation, the gIII-specific proteins were solubilized in sodium dodecyl sulfate (SDS) sample buffer and electrophoresed in a SDS-10% polyacrylamide gel (Fig. 3A). The rate at which the 74-kilodalton (kDa) gIII precursor was processed to the 92-kDa mature form was determined for each virus infection by densitometry by using an LKB 2222-020 Ultroscan XL laser densitometer (LKB Instruments, Inc.) (Fig. 3B) to scan the autoradiographs. Compar-
A Chase TiEmTi
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FIG. 3. (A) Pulse-chase analysis of gIII expressed from PRV-Be or PRV1001. PK15 cells (infected at a multiplicity of infection of 10) were pulse-labeled at 6 h postinfection with [35S]cysteine for 2 min and then incubated in the presence of excess nonradioactive cysteine and methionine. At the chase times indicated (in minutes) above each lane, the infected cell and medium fractions were harvested and the gIll species were immunoprecipitated by using 282 serum. Immunoprecipitations were then resolved on a SDS-10% polyacrylamide gel and visualized by fluorography. The infecting viral strain is indicated at the left of each panel (Be, 1001, 1002, 1003). The 74-kDa (precursor) and 92-kDa (mature) forms of gIII are indicated. (B) To quantitate the amounts of precursor and mature forms of gIll at each time point in the pulse-chase experiment, the autoradiographs were scanned by densitometry. The percentage of mature gIll present at each time point was calculated by using the following equation: (total 92 kDa/total 74 kDa + 92 kDa) x 100. The 180-min time points which were included in the quantitation graph are not shown in Fig. 3A. The curve corresponding to each of the virus strains is indicated. ison of PRVBe with PRV1001 indicates that the conversion of the BamHI site in the wild-type gIII gene to an EcoRI site had no effect on the kinetics of posttranslational processing or the stability of the gIII protein. It is also clear that PRV1002 and PRV1003 are indistinquishable from PRVBe or PRV1001 by this analysis. Furthermore, analysis of the medium fraction indicated that neither of the mutant proteins produced from cells infected with PRV1002 or PRV1003 was released into the medium (data not shown). Thus, a gIII protein lacking the last nine amino acids encoding the putative cytoplasmic domain or one with a specific change of Cys-471 to serine is synthesized and exported normally. Localization of gIII proteins with mutations in the cytoplasmic domain. Infected cultures were radiolabeled continuously for 16 h with [3Hlglucosamine and then separated into three fractions comprised of infected cells washed free of extracellular virions, virions released into the medium, or
VOL. 64, 1990
NOTES
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FIG. 4. Localization of wild-type and mutant forms of glycoprotein gIll during steady-state radiolabeling conditions. PK15 cells infected at a multiplicity of infection of 10 with parental or virus and labeled with [3H]glucosamine for 16 h. The cultures were divided into cell, virion, and medium fractions as described in the text, and representative amounts of each fraction were used in an immunoprecipitation procedure with different antibodies. Immunoprecipitates were resolved on a SDS-10%o polyacrylamide gel, and the 3H-labeled proteins were visualized by fluorography. Fractions are indicated at the upper left: cells (A), virions (B), and medium (C). The antibodies used are indicated across the top: anti-gIll goat polyclonal, 282 boiled or 282; anti-gIll monoclonal, Ml; and anti-gIll polyclonal, 284. The infecting virus strains are indicated: Be (PRV-Be), 01 (PRV1001), 02 (PRV1002), and 03 (PRV1003). A control for secretion is included in panel C and is designated 59 (PRV59). The positions of molecular mass standards (in kilodaltons) are shown at right. were
mutant
medium depleted of cells and virions as described and referenced by Ryan et al. (15). The gIII-specific proteins were immunoprecipitated from each fraction with either the
anti-gIII monoclonal antibody Ml, which primarily
recog-
nizes the mature form of the protein, or the polyclonal anti-gIII serum 282, which recognizes both precursor and mature forms of gIII. As a control for determining the relative efficiency of infection, the unrelated envelope glycoprotein gIl was immunoprecipitated from each fraction by using the polyclonal anti-gIl serum 284. Each of the immunoprecipitated fractions was analyzed on a SDS-10% polyacrylamide gel and visualized by fluorography (Fig. 4). Within infected cells (Fig. 4A), both forms of the gIII glycoprotein were immunoprecipitated with the polyclonal anti-gIII serum 282. Furthermore, boiling the infected cell fractions prior to adding the 282 serum significantly enhanced immunoprecipitation of the mature form of gIII. The
3519
electrophoretic mobility of the two forms of the gIll protein produced from cells infected with PRV1003 (cysteine to serine) was identical to that produced from cells infected with either PRVBe or PRV1001. The mature and precursor forms of gIll produced from cells infected with PRV1002 (cysteine to amber) exhibited an expected increase in mobility due to the deletion of nine amino acids from the C terminus of the protein (Fig. 4A, 282, lane 02). By using the amount of gIl immunoprecipitated with the 284 serum from the same infected cell extracts to control for differences in the efficiency of virus infection (Fig. 4A, 284), we can conclude that a comparable amount of both the precursor (74-kDa) and the mature (92-kDa) form of the gIII protein was present in each infected cell culture (Fig. 4A, 282 boiled, 282, and Ml). These results indicate that altering the proposed cytoplasmic domain of gIII has no apparent effect on the level of expression of the protein within infected cells. Though both precursor and mature forms of gIll were detected in cells infected with PRVBe, PRV1001, PRV1002, or PRV1003, only the larger 92-kDa mature form of the glycoprotein was expected to be found in virions (15). Total virion and immunoprecipitated gIl profiles indicate that the infections had progressed to a similar extent (Fig. 4B, total virions and 284). Immunoprecipitation of gIll from these virions indicated that similar quantities of the mature 92-kDa gIII were present (Fig. 4B, 282). These results indicate that changing Cys-471 to serine or to a nonsense codon that effectively removes the putative cytoplasmic tail of gIII has no effect on the ability of the mutant proteins to be incorporated into virions. Significantly, neither the gIII protein nor the control gII protein was detected in the media obtained from cultures infected with either the parental or mutant viral stocks (Fig. 4C). This is in contrast to the recombinant virus PRV59 (unpublished data), which contains a linker insertion at the BamHI site within the gIII carboxy-terminal hydrophobic domain, resulting in the secretion of the gIII protein into the medium (Fig. 4C, lane 59). These results, in conjunction with our previous observation that the recombinant viruses expressing the mutant gIII proteins reacted with the Ml antibody at the cell surface in the black-plaque assay, indicate that the cytoplasmic tail mutations do not significantly alter the ability of the gIII protein to remain stably anchored at the cell surface. In summary, pulse-chase analysis of PK15 cells infected with either PRV1002 (Cys-471 to amber) or PRV1003 (Cys471 to serine) indicated that both the rate of processing and the efficiency of export of the precursor form to the mature form of the mutant gIll proteins were not significantly different from those of the parental glycoprotein. Steadystate labeling experiments of cells infected with the mutant viruses provided evidence for normal levels of cellular expression of the mutant gIII proteins. These results indicated that the putative cytoplasmic domain of gIII is not required for normal cellular processing and localization. Evidence that the mutant gIII proteins were stably anchored within membranes was provided by three sets of data. Purified virions from cells infected with PRVBe, PRV1001, PRV1002, or PRV1003 contain comparable amounts of gIII in the virus envelope. The medium fractions obtained from infected cells, labeled by either the pulsechase or steady-state format, did not contain detectable quantities of the gIII mutant proteins. Both the PRV1002 and PRV1003 recombinant viruses produced plaques which reacted with the Ml antibody in the black-plaque assay, indicating that the mutant gIII proteins are at the cell surface
3520
J. VIROL.
NOTES
(4). These data, collectively, indicate that removing the cytoplasmic domain from gIll or changing the cysteine to a serine had no effect on the ability of the gIll protein to be assembled into virions or stably anchored at the cell surface. This conclusion differs from that made by Holland and co-workers (6), who performed similar experiments testing the function of the HSV-1 gC cytoplasmic domain. The predicted cytoplasmic domain of HSV-1 gC is similar to that of gIII with respect to size but differs with respect to the number of positively charged amino acids present within this region of the proteins. PRV gIll has only one positively charged residue, while HSV-1 gC has five. The cytoplasmic domains of proteins known to span membranes typically contain several positively charged amino acids and, for some proteins, it is thought that these charged residues may play a role in the retention of the proteins within membranes (3, 6, 11). Holland et al. found that deletion of 14 amino acids, including the entire 11-amino-acid cytoplasmic domain from the HSV-1 gC glycoprotein, resulted in normal synthesis, export, and transport of the protein to the cell surface (6). However, unlike PRV gIll mutants, the mutant gC protein was released slowly into the medium of infected cells. This phenotype may not be solely due to the absence of the hydrophilic tail, since the hydrophobic transmembrane domain of gC was also shortened by three amino acids in this mutant. It is clear, however, that the last 14 amino acids of gC are required for stable anchoring of the protein in the membrane. The apparent difference in membrane retention between the gC and gIll mutant proteins lacking their respective cytoplasmic domains may be attributed to the difference in the number of positive charges present within these domains. Thus, the positively charged residues found in most membrane-spanning proteins may ensure membrane retention, whereas proteins like gIll, which contain very few charged residues, may depend on other amino acids within the cytoplasmic domain or even other parts of the protein to ensure membrane retention. We would expect that if this idea holds true, the cytoplasmic domain of the VZV gpV protein would not be required for membrane retention since, like PRV gIll, it has only one positively charged amino acid in this region (Fig. 1B). Although we did not specifically change the single positively charged arginine residue at position 475, it is clear from our analysis of PRV1002 (cysteine to amber) that gIII does not require this charged amino acid or any other cytoplasmic domain amino acid for membrane retention. From these results, it is likely that efficient membrane anchoring of gIll depends only on the presence of the postulated hydrophobic transmembrane domain. It is also possible that the retention of gIll in membranes could depend upon interactions between a cellular or viral component and regions of the gIll glycoprotein other than the cytoplasmic domain. Further characterization of the gIll protein and its potential interaction with other membrane proteins is currently under way to test these possibilities. The presence of the conserved cysteine residue (Cys-471) in five of the seven members of the gC gene family was of some interest. Initially, we thought that fatty acid modification of this residue, as noted for other membrane proteins, may be a possibility (9, 14, 18). However, as yet, we have no direct evidence for acylation of gIII (unpublished observations). There was no detectable phenotype for a glll protein carrying a serine at position 471 rather than a cysteine. A role for the sulfhydryl functionality of cysteine in potential disulfide bond formation is also difficult to determine, since changing Cys-471 to a nonsense codon or a serine codon
gave rise to a gIII protein indistinguishable by our techniques in export and localization from parental gIII. The role, if any, of this conserved cysteine in the gC family remains to be determined. Virion envelopes are typically devoid of host proteins, which suggests that inclusion of a protein into a virus particle might depend on a specific signal for assembly. It has been postulated that the signals responsible for incorporation of a protein into virions may be contained within the cytoplasmic domain of a membrane-spanning protein (for reviews, see references 19 and 22). This certainly appears to be the case for some RNA-enveloped viruses. For example, Semliki Forest virus assembly is mediated by the interaction of the cytoplasmic domain of the E2 spike glycoprotein with the viral capsid protein (24). This model does not seem to apply to PRV gIII or to HSV-1 gC. Our work and that of Holland et al. (6) suggest that absence of the putative cytoplasmic domain has no effect on the ability of the protein to be assembled into virions. These results are similar to those obtained by Perez et al. (11) with RSV Env protein lacking the cytoplasmic domain. It is clear that mechanisms other than interaction at the carboxy terminus must exist for the assembly of PRV gIII, HSV-1 gC, and, most likely, the other members of the gC family into virions. We thank Nels Pederson for help in designing the mutagenic primers and noticing that the BamHI site could be changed to an EcoRI site without affecting the protein. We also thank Daniel Tenney and Patrick Ryan for critical reviews of the manuscript.
1. 2. 3. 4.
5.
6. 7.
LITERATURE CITED Ben-Porat, T., J. M. DeMarchi, and A. S. Kaplan. 1974. Characterization of defective interfering viral particles present in a population of pseudorabies virions. Virology 61:29-37. Ben-Porat, T., and A. S. Kaplan. 1985. Molecular biology of pseudorabies virus, p. 105-173. In B. Roizman (ed.), The herpesviruses. Plenum Publishing Corp., New York. Cutler, D. F., and H. Garoff. 1986. Mutants of the membranebinding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity. J. Cell Biol. 102:899-901. Enquist, L. W., C. L. Keeler, Jr., A. K. Robbins, J. P. Ryan, and M. E. Whealy. 1988. An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation. J. Virol. 62:3565-3573. Fitzpatrick, D. R., L. A. Babiuk, and T. J. Zamb. 1989. Nucleotide sequence of bovine herpesvirus type 1 glycoprotein gIll, a structural model for gIll as a new member of the immunoglobulin superfamily, and implications for the homologous glycoproteins of other herpesviruses. Virology 173:1-12. Holland, T. C., R. J. Lerch, and K. Earhart. 1988. The cytoplasmic domain of herpes simplex virus type 1 glycoprotein C is required for membrane anchoring. J. Virol. 62:1753-1761. Holland, T. C., R. M. Sandri-Goldin, L. E. Holland, S. D. Marlin, M. Levine, and J. C. Glorioso. 1983. Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay. J. Virol. 46:649-652.
8. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82:488-492. 9. Mack, D., and J. Kruppa. 1988. Fatty acid acylation at the single
cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus. Biochem. J. 256:1021-1027. 10. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11. Perez, L. G., G. L. Davis, and E. Hunter. 1987. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intra-
NOTES
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cellular transport and assembly into virions. J. Virol. 61:29812988. Robbins, A. K., R. J. Watson, M. E. Whealy, W. W. Hays, and L. W. Enquist. 1986. Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C. J. Virol. 58:339-347. Robbins, A. K., M. E. Whealy, R. J. Watson, and L. W. Enquist. 1986. Pseudorabies virus gene encoding glycoprotein glll is not essential for growth in tissue culture. J. Virol. 59:635-645. Rose, J. K., G. A. Adams, and C. J. Gallione. 1984. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc. Natl. Acad. Sci. USA 81:2050-2054. Ryan, J. P., M. E. Whealy, A. K. Robbins, and L. W. Enquist. 1987. Analysis of pseudorabies virus glycoprotein gIlI localization and modification by using novel infectious viral mutants carrying unique EcoRI sites. J. Virol. 61:2962-2972. Ryan, J. P., M. E. Whealy, A. K. Robbins, C. L. Keeler, Jr., and L. W. Enquist. 1989. Genetic analysis of a herpesvirus glycoprotein signal sequence, p. 299-308. In R. W. Compans et al. (ed.), Cell biology of virus entry, replication, and pathogenesis. Alan R. Liss, Inc., New York. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. Schmidt, M. F. G. 1982. Acylation of proteins-a new type of
19. 20.
21. 22. 23.
24.
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