of Vero Cells - Journal of Virology - American Society for Microbiology

Vol. 67, No. 5

JOURNAL OF VIROLOGY, May 1993, p. 2646-2654

0022-538X/93/052646-09$02.00/0 Copyright © 1993, American Society for Microbiology

The Amino-Terminal One-Third of Pseudorabies Virus Glycoprotein glll Contains a Functional Attachment Domain, but This Domain Is Not Required for the Efficient Penetration of Vero Cells SHANNON J. FLYNN,' BRADFORD L. BURGETT,' DANIEL S. STEIN,2t KIMBERLY S. WILKINSON,1 AND PATRICK RYAN'*

Department of Microbiology and Immunology' and Department of Medicine,2 University of Tennessee, Memphis, Memphis, Tennessee 38163 Received 28 December 1992/Accepted 20 January 1993

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIll were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4°C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIll is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances. The envelope of pseudorabies virus (PRV) contains at least six glycoproteins (12, 20-23). Of these, glycoprotein gIll, homologous to herpes simplex virus (HSV) gC, is perhaps the best characterized. DNA sequence analysis of the gIII gene predicts a 479-amino-acid product that contains eight N-linked glycosylation sites (23). Mature wild-type gIII can be found in the virus envelope and at the infected cell surface as a 92-kDa protein, having inserted into membranes by using the host cell's exocytic pathway (26). PRV strains lacking gIII are infectious but have been described as defective in attachment to and penetration of target cells (15, 24, 25, 28, 37). Additionally, virions lacking gIII and a second glycoprotein, gI, are released poorly from infected cells (28). From these studies of null mutants, it has been concluded that gIII is nonessential but multifunctional (28). Still, to date only two functional domains have been experimentally assigned to structural regions. The gIII signal sequence lies within amino acids 1 to 22 and is required for insertion of the glycoprotein into cell membranes and the virus envelope (3). Once inserted, the stable anchoring of gIII in these membranes is dependent on a membranespanning region composed of amino acids 436 to 470 (29). Thus, only functions involved in the proper localization of gIII have been mapped. Here, we report on the preliminary mapping of another functional domain in gIII. Mettenleiter et al. (17) have demonstrated that gIII functions as a PRV receptor by interacting with cellular heparinlike substances; an analo-

situation has been shown for members of the gC family in HSV and bovine herpesvirus (7, 19). The cellular component appears to be heparan sulfate, a glucosaminoglycan which, when associated with protein, forms a common proteoglycan found on cell surfaces (11). As a population, heparan sulfate can be considered to have an extremely heterogeneous distribution of negative charge, the result of an imprecise mechanism that places sulfates at various positions on its repeating disaccharide units (13). Accordingly, PRV initially binds to target cells through an electrostatic interaction presumably mediated by positive charges found on gIII and the negatively charged backbone of heparan sulfate. As suggested for other herpesviruses (5), this interaction is generally regarded as a necessary prelude to efficient virus entry via a cascade of events that includes secondary attachment to an unidentified cellular receptor (10) and culminates in a pH-independent fusion of the virus envelope with the plasma membrane (34). We have introduced a set of defined deletions into the PRV genome that essentially divide gIll into three structural domains of equal size. We then determined whether any of the adjacent or overlapping deletions functionally removed the gIII attachment domain. Our results indicated that the amino-terminal one-third of gIII contains a functional attachment domain for PK15 and Vero cells. However, the binding brought about by this domain was required only for the timely penetration of PK15 cells. Strains that lack the first one-third of gIII and are therefore attachment deficient still penetrated Vero cells efficiently. Thus, initial virus attachment to cells via a gIII-heparan sulfate interaction is not a prerequisite for efficient virus entry of all cell types. gous

* Corresponding author. t Present address: National Institute of Allergy and Infectious Diseases, Rockville, MD 20852.

2646

VOL. 67, 1993

ATTACHMENT MUTANTS OF PRV gIII MATERIALS AND METHODS

Cells, virus, and DNA. PK15 (porcine kidney) and Vero (African green monkey kidney) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as described previously (26). The Becker strain of PRV (PRVBe), PRV2, PRV500, and PRV501 have been described previously (25, 26) and were propagated on PK15 cells. Escherichia coli KK2186 is isogenic to JM103 (14) except that it lacks a cryptic P1 prophage. Our plasmid constructs were made by standard recombinant DNA techniques (14) and verified by restriction enzyme analysis and dideoxy DNA sequencing. Mutagenesis of the gIII gene. All deletions were first introduced into plasmid clones of gIII prior to recombination into the virus genome. Codons 2 to 458 of gIII were deleted by ligating EcoRI-PstI fragments from plasmids pIG504 and pKS1001 (plasmid pIG504 is a derivative of pIG5-; pIG5- is identical to pALM15 [24] except that it contains the IG region of phage M13 and lacks any EcoRI sites). Plasmid pIG504 contains a deletion of codons 2 to 292 of gIII and has instead a unique EcoRI site that immediately follows the initial ATG codon of gIII. Plasmid pKS1001 has been described previously (30) and contains a unique EcoRI site that has replaced the BamHI site found near the 3' end of the wild-type gIII coding sequence (see Fig. 1). Each of these plasmids was digested with restriction enzymes EcoRI and PstI, with the latter enzyme cutting at a unique site in the ampicillin resistance gene of each plasmid. A 2.4-kbp fragment containing the first codon of gIII and about 1 kbp of upstream PRV DNA was isolated from pIG504, and a 3.7-kbp fragment containing the final 21 codons of gIII and about 1 kbp of downstream PRV DNA was isolated from pKS1001. After ligation and transformation of competent cells of KK2186 (selecting ampicillin-resistant colonies), a plasmid designated pIG509 that had a unique EcoRI site in place of codons 2 to 458 of gIIn was identified. No additional codons were introduced in this construct. To delete codons 25 to 157 of gIII, pIG5- was partially digested with restriction enzymes SalI and Sac. The digested DNA was religated in the presence of a SalI-SphISacl linker (see below), digested with SphI to remove any multiple insertions of the linker, religated, and then used to transform competent cells of KK2186. Among the transformants, a plasmid that contained a deletion starting 3' to the SalI site at the end of the signal sequence coding region and ending at the first Sacl site in the gIII gene (Fig. 1) was identified and designated pIG523. The missing sequence was replaced with the following linker: 24 Ser Met Gln (158) 5'-TCG AQC ATG CAG CT-3' 3'-CG TAC G-5' This linker introduced a unique SphlI site (underlined) and three new codons resulting in insertion of the amino acid sequence Ser-Met-Gln (indicated above the linker sequence) between positions 24 and 158 of the wild-type sequence. Codons 265 to 411 of gIII were deleted by exonuclease digestion. Plasmid pIG3 contains an in-frame fusion of gIII to the E. coli lacZ gene; the fusion joint lies at the BamHI site near the 3' end of gIII (Fig. 1). Because the hybrid gene can be expressed from an E. coli tac promoter, colonies synthesizing in-frame fusion proteins are blue on medium containing X-Gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside) (14). Plasmid pIG3 was linearized at a unique

2647

KjpnI site

at codons 330 and 331 of gIII and digested with Bal3l exonuclease. The digested DNA was ligated and used to transform competent cells of KK2186. Ampicillin-resis-

tant blue colonies were obtained, and their plasmids were screened for deletions within gIII. The exact endpoints of these deletions were determined by DNA sequence analysis, and one was found to be a deletion of codons 265 to 411. The lacZ portion of the plasmid harboring this deletion was subsequently replaced with PRV DNA that normally lies downstream of the BamHI site and that is found in pIG5The resulting plasmid was designated pIG505. Construction and identification of PRV mutants. The recombinant viruses PRV505, -509, and -523 were generated by cotransfection of plasmid DNA and virus genomic DNA by calcium phosphate methods described previously (6). Specifically, pIG509 was cotransfected with PRV-Be genomic DNA to generate PRV509. The black-plaque assay (9) was used to identify PRV509 candidates because the correct recombinants did not express gIII on the infected cell surface and were therefore nonreactive to polyvalent goat antiserum 282 (26) that is directed against gIII. PRV-Be plaques did bind this antibody and were therefore stained black by a second, peroxidase-linked goat antibody in the presence of 4-chloro-1-naphthol. Under these conditions, PRV509 plaques remained unstained. The genomic DNA of PRV10, a strain deleted for the gIII promoter and 87% of its coding sequence (24), was cotransfected with pIG505 to generate PRV505, and DNA from PRV509 was cotransfected with pIG523 to generate PRV523. Again, recombinant viruses were identified by the black-plaque assay because each expressed gIII on the infected cell surface while the parent strains PRV10 and PRV509 did not. Virus constructs were verified by Southern blot analysis of viral DNA and by analysis of radiolabeled gIII that was immunoprecipitated from either purified virions or infected cells as described previously (24, 26). The immunoprecipitated forms of gIII were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by fluorography and autoradiography (26). Attachment assay. Attachment assays were performed in triplicate. Monolayers of PK15 or Vero cells on 60-mm dishes were inoculated at 4°C with diluted stocks of wildtype or mutant virus containing approximately 150 PFU. After 2 h, the inocula were removed and control monolayers were immediately overlaid with DMEM supplemented with 2% fetal bovine serum and 1% methylcellulose (methylcellulose medium). After removal of the inoculum, the experimental monolayers were washed twice vigorously with icecold phosphate-buffered saline (PBS) and then overlaid with methylcellulose medium. The infected monolayers were then incubated for 36 h at 37°C to allow plaque formation. The average number of plaques per plate on PBS-washed monolayers was determined, divided by the average number of plaques per plate on untreated control monolayers infected with the same strain, and multiplied by 100 to represent relative attachment efficiency as a percentage of the control value. Penetration assay. Penetration assays were performed in triplicate. Monolayers of PK15 or Vero cells on 60-mm dishes were inoculated at 37°C with diluted stocks of wildtype or mutant virus containing approximately 150 PFU. After 1 h of incubation the inocula were removed and the infected monolayers were either (i) immediately overlaid with methylcellulose medium, (ii) washed twice with PBS (at room temperature) and then overlaid with methylcellulose medium, or (iii) overlaid at room temperature with 2 ml of

2648

J. VIROL.

FLYNN ET AL.

A Be 509 523 505 P PB P PB P PB P PB

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FIG. 1. Characterization of gIll deletion strains. (A) Southern blot of PRV-Be, PRV509, PRV523, and PRV505 genomic DNAs. Viral DNAs were digested with PstI (P) or PstI and BseAI (P/B), electrophoresed, transferred to nitrocellulose, and probed with 35S-labeled pGBe4.3, a derivative of pGEM-3Zf(+) (Promega Corp.) that has inserted into it a 4.3-kbp PstI fragment containing gIII. Migration of molecular mass markers is shown at the left (in kilobase pairs), and the strain designations are indicated at the top. (B) Genomic location of a 4.3-kbp PstI fragment of PRV that carries gIII. The PRV genome is depicted, and the unique long (UL), the unique short (Us), and the inverted and terminal repeats (IRS and TRs, respectively) are indicated. The PstI fragment has been expanded and sized in kilobase-pair increments and is shown as a restriction map indicating the positions of the relevant restriction sites. The open arrow designates the limits and direction of transcription of the gIII gene. (C) Depiction of the wild-type glll protein made by PRV-Be and the gIII deletions found in mutant strains. The black boxes shown at either end of the 479-amino-acid gIII polypeptide represent the signal sequence at the amino terminus and the transmembrane anchor near the carboxy terminus; N's designate the position of N-linked glycosylation sites in gIII. The relative sizes and positions of the deletions within gIll are shown by the hatched bars. The amino acids deleted are indicated within each bar. Strain designations are indicated at the left.

citrate buffer (40 mM sodium citrate, 10 mM KCl, 135 mM NaCl [pH 3.0]) (8). After 2 min, the citrate buffer was removed and the monolayers were washed twice with PBS and then overlaid with methylcellulose medium. The infected monolayers were then incubated for 36 h at 37°C to allow plaque formation. The average number of plaques per plate on PBS-washed and citrate-treated monolayers was determined and independently divided by the average number of plaques per plate on untreated control monolayers infected with the same strain. When multiplied by 100, the values for the PBS-washed plates represented relative attachment and penetration efficiencies combined, while the values for the citrate-treated monolayers represented relative penetration efficiency alone, both as a percentage of the control value.

Attachment in the presence of heparin. Heparin competition experiments were performed in triplicate. This assay was performed as described earlier for the attachment assay except that virus stocks were diluted in medium containing 0, 0.01, 0.1, 0.5, 1.0, or 5.0 ,ug of heparin (Sigma Chemical Co.) per ml. The average number of plaques per plate from heparin-treated infections was determined, divided by the average number of plaques per plate on untreated control monolayers infected with the same strain, and then multiplied by 100 to represent relative heparin resistance as a percentage of the control value. Attachment assay with heparinase-treated cells. Attachment experiments were performed in triplicate. Monolayers of PK15 or Vero cells on six-well dishes were incubated with PBS-1 mM CaCl2 or PBS-1 mM CaCl2 containing 1 U of

ATTACHMENT MUTANTS OF PRV gIll

VOL. 67, 1993

heparinase (Boehringer Mannheim Corp.) per ml for 1 h at 37°C. The monolayers were then washed three times with ice-cold PBS, inoculated with approximately 100 PFU of PRV-Be, PRV509, or PRV523, and incubated at 4°C for 2 h. The infected monolayers were subsequently washed twice with ice-cold PBS and overlaid with methylcellulose medium. The monolayers were then incubated at 37°C for 36 h to allow plaque formation. The average number of plaques per well on heparinase-treated monolayers was determined, divided by the average number of plaques per well counted on the untreated control monolayers infected with the same strain, and then multiplied by 100 to represent relative attachment efficiency on heparinase-treated cells as a percentage of the control value. RESULTS

Construction and characterization of PRV gIll mutants. Previous studies have shown that virions completely devoid of gIII in their envelopes do not attach efficiently to cultured cells (25, 28, 37). To more specifically determine that portion of gIII involved in virus attachment, three gIII deletions were introduced in plasmid copies of wild-type gIII and subsequently recombined into isogenic virus backgrounds to construct strains PRV509, -523, and -505 (see Materials and Methods). The gIII allele of PRV509 contains an in-frame deletion of codons 2 to 458 and therefore encodes only the final 21 amino acids of the wild-type protein. This strain effectively represents a gIII null mutant. Strain PRV523 is deleted for gIII codons 25 to 157 and thus produces a gIII polypeptide containing a functional signal sequence (ensuring proper localization [3]) but otherwise lacking the first one-third of the full-length protein. Strain PRV505 harbors a deletion largely confined to the final one-third of the gIII gene. The loss of codons 265 to 411, however, does not remove the transmembrane anchor sequence (30). Consequently, deletion analyses with strains PRV523 and PRV505 in conjunction with the previously described PRV2, which contains an in-frame deletion of the middle one-third of gIII (26), allowed us to divide the gIII protein into three structural domains of nearly equivalent sizes. The extent of each of these deletions with respect to the wild-type gIII protein is indicated in Fig. 1C. A Southern analysis of viral DNAs from PRV509, PRV523, and PRV505 was performed with a gIII-specific probe to confirm that each intended deletion was correctly crossed into the virus genome (Fig. 1A). As noted previously, the gIII gene of PRV-Be, our wild-type strain, lies on a 4.3-kbp PstI fragment (24). As indicated in Fig. 1B, this fragment can be further cleaved into a left-hand 2.3-kbp fragment and a right-hand 2.0-kbp fragment when digested with the restriction enzyme BseAI, which has a single recognition site midway through the gIII coding sequence. As expected, the deletion contained within PRV509 eliminated the BseAI site and reduced the PstI fragment by nearly 1.4 kbp (Fig. 1A, lanes P and P/B under 509). The deletions introduced into strains PRV523 and PRV505 also reduced the length of the PstI fragment, each by about 400 to 450 bp (Fig. 1A, lanes P under 523 and 505). Double digests of viral DNAs were performed with restriction enzymes PstI and BseAI to more specifically map the deletions in these strains. As shown in Fig. 1A (lane P/B under 523), the intended 405-bp deletion in PRV523 altered the migration of the left-hand PstI-BseAI fragment so that it was no longer resolved from the right-hand BseAI-PstI fragment that was unaffected by the deletion. Conversely,

599g

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2649

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FIG. 2. virus envelopes. PK15 cells were infected with either wild-type or mutant virus (indicated at the top) at a multiplicity of infection of 10 and incubated at 37°C for 16 h in the presence of [3H]glucosamine. After each cell culture medium had been harvested, virions were purified as described in Materials and Methods. gIII and gII were immunoprecipitated with goat polyclonal antisera 282 (26) and 284 (33), respectively, as described in Materials and Methods. The immunoprecipitates were then resolved by SDS-PAGE and fluorography and visualized by autoradiography. Lanes containing gIII and gII immunoprecipitates are indicated by III and II, respectively, at the top of each lane. Positions of molecular mass markers are indicated at the left (in kilodaltons). Only the relevant portion of the autoradiogram is shown.

the resolution of the two PstI-BseAI fragments was increased in DNA from PRV505 because the intended deletion of 450 bp was confined to the smaller, right-hand fragment (Fig. 1A, lane P/B under 505). Each of the mutant strains PRV523, PRV2, and PRV505 contains glIl in the virus envelope. Because proteins that are depleted of major segments are often misfolded and consequently incorrectly localized, we needed to confirm that the altered gIII proteins encoded by PRV523 and PRV505 were still successfully incorporated into virus envelopes after infection of cultured cells. The correct localization of the PRV2 gIII product to the virus envelope has been shown previously (26). To determine the gIII content of each mutant virus's envelope, [3H]glucosamine-labeled gIII was immunoprecipitated from purified, mature virions of each strain as described previously (26). Additionally, a second glycoprotein, gIl, that was unaltered in each mutant strain was immunoprecipitated as a measure of the level of [3H]glucosamine incorporation that was achieved with each infection. The immunoprecipitates were resolved on an SDSpolyacrylamide gel and subjected to autoradiography to visualize the bands. Glycoprotein gIII was found in the virus envelope of strains PRV-Be, PRV523, PRV2, and PRV505, but, because of their deletions, the mutant glycoproteins migrated differently from wild-type gIII in the polyacrylamide gel (Fig. 2). Note that the immunoprecipitation from the null mutant PRV509 indicated that no cross-reactive species comigrated with the wild-type or any of the mutant forms of gIll. It was independently determined that PRV505 contained only about half of the wild-type level of gIII in its envelope (31). While not readily apparent in Fig. 2, it is quite likely that PRV523 and PRV2 also produced virions with diminished amounts of gIII in their envelopes as a result of somewhat impaired export of the mutant gIII species in infected cells (4, 26). Because of the smaller amount of gIII in some of the mutant virions, we included two additional PRV mutants with quantitated reductions in the level of gIII in their envelopes. Strains PRV500 and PRV501 contain mutations in the signal sequence coding region of gIII (25, 27). Consequently, only 40% of the PRV500 gIII products and 15% of the PRV5O1 gIII products are translocated across

J. VIROL.

FLYNN ET AL.

2650

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509

523

2

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500

501

FIG. 4. Attachment and penetration profiles of PRV-Be and mutant strains on Vero cells. See the legend to Fig. 3.

were performed as described in Materials and Methods. (A) Relative

attachment efficiency of each strain after 1 h at

4°C.

(B) Relative

absorption and penetration efficiency of each strain after 1 h at

37°C.

They axis in each graph is the average PFU per plate, expressed as a percentage of the control values.

Strain designations are given

along the bottom. The number at the top of each bar is the average number of PFU per plate (as a percentage of the control value) of

that particular strain that were (A) resistant to PBS washing at or (B) resistant to PBS washing at

treatment at

37°C

the rough

endoplasmic

37°C

4°C

(shaded bars) or to citrate

(hatched bars).

membrane;

reticulum

the balance

remains in the cytosol of the infected cell. This leads to a similar reduction in

gIII

levels in the envelopes of PRV500

and PRV5O1 virions, as shown in Fig. 2. Deletion of amino-terminal one-third of gIll

results in an

attachment defect on PK15 cells similar to that observed for viruses completely lacking gIll virus penetration

in their envelopes. Because

does not occur at low temperature,

the

efficiency of virus attachment can be shown experimentally by determining the percentage of total input PFU that are not removed from infected cell surfaces by vigorous washing with PBS after an initial 2-h adsorption period at

4°C

(15).

We have used this experimental design (see Materials and Methods) mutant

to

evaluate

strains;

the

proficiency

attachment

the

results

are

summarized

in

of our

Fig.

3A.

Although none of the mutant strains was as resistant to PBS

treatment as PRV-Be, neither a loss of the carboxyl-terminal two-thirds of gIll (represented by strains PRV2 and PRV5O5, collectively) nor a significant reduction

in the

amount of

full-length gIll in the virus envelope (represented by PRV500 and

PRV5O1)

efficiently

to

deprived PK15

the

cells.

virus

its

of

Only when

ability

the

to

attach

amino-terminal

one-third of gIll was deleted, as in PRV523 virus envelopes, did the virus become as sensitive to PBS washes as the null mutant PRV5O9. Each of these viruses had nearly 80% or more of their input PFU removed by washing.

This sug-

gIII

possessed

gested, therefore, that the first one-third of

the primary attachment domain of PRV and that this domain functioned independently of its levels in the virus envelope. Because it has been reported that

gIII

plays an indirect

role in virus penetration (15, 36), we also determined the penetration efficiency of each of our strains. To do so, monolayers of PK15 cells were overlaid with wild-type or mutant virus at 37°C. After 1 h, the monolayers were (i) left untreated, (ii) washed twice with PBS, or (iii) exposed to citrate buffer (pH 3.0) for 2 min (see Materials and Methods). Monolayers treated with acid pH citrate buffer were subsequently washed twice with PBS before all monolayers were overlaid with methylcellulose medium and incubated at 37°C to allow plaque formation. Citrate buffer (pH 3.0) inactivates virus particles that are present on the cell surface but not those that have fused with the plasma membrane. Therefore, resistance to citrate treatment indicates virus penetration (8). The results of our penetration experiments mirrored those obtained in the 4°C attachment assay. The number of PFU that were resistant to PBS treatment alone (Fig. 3B, shaded bars) was generally about the same as or slightly higher than that determined at 4°C. Focusing on the citrate-treated monolayers (Fig. 3B, hatched bars), greater than 50% of the control number of input PFU had penetrated cells by 1 h postinfection for PRV-Be and each of the mutants determined to be attachment proficient at 4°C. Only with the null mutant PRV5O9 and the attachment-defective mutant PRV523 had fewer than 20% of the control number of virions penetrated by 1 h. Thus, in agreement with results obtained by others (15, 36), we observed a direct correlation between attachment proficiency and penetration proficiency on PK15 cells. Efficient penetration of Vero cells does not require gIIImediated attachment. To determine whether the attachment and penetration phenotypes shown in Fig. 3 could be obtained on another cell type, we repeated our assays on Vero cells. Profiles almost identical to those observed on PK15 cells were obtained for attachment to Vero cells at 4°C for all of the strains (Fig. 4A). Strains PRV5O9 and PRV523 appeared to be slightly more resistant to PBS treatment on Vero cells than PK15 cells but clearly remained attachment deficient.

VOL. 67, 1993

Virus penetration of Vero cells was markedly different from penetration of PK15 cells. In contrast to the results obtained at 4°C, all of the strains, including PRV509 and PRV523, were resistant to PBS treatment on Vero cells at 37°C (Fig. 4B, shaded bars). For PRV509 and PRV523, this resistance was due not to efficient attachment but rather to efficient penetration. In fact, all of the mutant strains penetrated Vero cells efficiently, with nearly 50% or more of the control number of input PFU becoming resistant to citrate treatment by 1 h postinfection (Fig. 4B, hatched bars). These results demonstrated that efficient primary attachment is not necessarily required for rapid virus penetration. Contributing to the significance of our results with gIII mutants was our finding that PRV-Be exhibited a penetration-deficient phenotype on Vero cells. Although PRV-Be attached efficiently at 37°C (Fig. 4B, shaded bar), only 14% of the control number of input PFU had penetrated the Vero cells by 1 h postinfection (Fig. 4B, hatched bar). Therefore, efficient attachment of wild-type virus did not appear to lead to productive penetration, even though mutant strains PRV2, -505, -500, and -501 exhibited both attachment- and penetration-proficient phenotypes on Vero cells. Our results suggested that abundant levels of full-length gIII in the virus envelope actually impeded the virus's entry into these cells. Binding of attachment domain mutants is relatively unaffected by soluble heparin compared with that of attachmentproficient strains. Previous studies have demonstrated that gIll mediates virus attachment by binding to cell surface heparan sulfate molecules (17, 37). Because of this interaction, wild-type PRV exhibits reduced infectivity in the presence of competing, exogenously added heparin, a glucosaminoglycan closely related to heparan sulfate (11, 17, 37). In contrast, mutant strains devoid of gIll are relatively resistant to heparin exposure (17, 37). The effect of heparin on the attachment of our wild-type and mutant strains was determined by inoculating monolayers of PK15 or Vero cells at 4°C in the presence of increasing concentrations of heparin and then vigorously washing the monolayers with PBS after 2 h. The infected monolayers were then allowed to form plaques at 37°C, and the number of plaques on each plate was compared with that in untreated control infections (see Materials and Methods). Each of the attachment-proficient strains PRV-Be, PRV2, and PRV505 was sensitive to incubation with heparin on both PK15 and Vero cells (Fig. 5). Productive attachment of virus particles was blocked 97% or more by the presence of 1 ,ug of heparin per ml on both PK15 and Vero cells. In contrast, neither PRV509 nor PRV523 was inhibited by more than 70% on either cell line at the highest concentration of heparin present. In other words, 10-fold more PFU were resistant to 5 ,ug of heparin per ml for PRV509 and PRV523 than for the attachment-proficient strains. Because of this relative immunity to heparin competition, it is likely that the virus attachment domain that is deleted in PRV523 functions as a heparin-binding site. Removal of heparan sulfate from cell surfaces does not interfere with adsorption of attachment domain mutants. Other investigators have specifically demonstrated the role of heparan sulfate molecules in herpesvirus infections by enzymatic removal of these moieties from cell surfaces (17-19, 35). We used heparinase to demonstrate that PRV523 infectivity is unaffected by the removal of heparan sulfate from the target cell's surface. Cell monolayers were treated with heparinase at 37°C (see Materials and Methods) prior to virus inoculation at 4°C. After 2 h, the monolayers were vigorously washed with PBS and then incubated at 37°C in

2651

ATTACHMENT MUTANTS OF PR, XIIl

0

1

2

3

4

5

6

Heparin (gg/mI) FIG. 5. Attachment of PRV-Be and mutant strains to (A) PK15 cells and (B) Vero cells in the presence of heparin. Assays were performed at 4'C as described in Materials and Methods. They axis, in logarithmic scale, is the average number of PFU per plate, expressed as a percentage of the control values. The x axis is the concentration of heparin used as a competitor. Symbols: El, PRVBe; x, PRV509; A, PRV523; 0, PRV2; *, PRV505. Note that control experiments were performed in which monolayers were incubated at 4°C with heparin (5.0 p.g/ml) for 2 h; the monolayers were subsequently washed vigorously twice with ice-cold PBS and then incubated for an additional 2 h with diluted virus in the absence of heparin before being overlaid with methylcellulose medium at 37°C. No significant reduction in infectivity was observed for any virus strain assayed (4).

methylcellulose medium to allow plaque formation. The results are depicted in Fig. 6. Compared with mock-digested cells, less than 20% of the PRV-Be input PFU attached to heparinase-treated PK15 cells (black bars) or heparinase-treated Vero cells (shaded bars). However, greater than 75% of the wash-resistant PFU of both PRV509 and PRV523 attached to heparinase-treated PK15 and Vero cells. Thus, wild-type PRV attachment was greatly reduced by the loss of heparan sulfate from the cell surface, but the mutants PRV509 and PRV523 were relatively unaffected by its absence. We conclude that these mutants are not dependent on heparan sulfate for productive infection of target cells, a conclusion reached by Mettenleiter et al. (17) with different PRV mutants completely lacking glll in their virions. We have described the rapid penetration of PRV509 and PRV523 virions into Vero cells in the absence of efficient virus attachment and, conversely, the retarded penetration of PRV-Be virions into Vero cells despite their ability to attach proficiently to these cells (Fig. 4). These results suggested that glll interfered with steps in PRV infection that occurred after the initial attachment to heparan sulfate. We performed an additional experiment with PRV-Be on heparinase-treated Vero cells in which the viral overlay was washed with PBS after a 1-h incubation at 37°C instead of 4°C (see Materials and Methods). These conditions assayed

2652

J. VIROL.

FLYNN ET AL. 120-

-c5~~~~~~~~~~N

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~

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0~~~~~~~0

1000

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0 IL

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40-

200

523 509 Be FIG. 6. Attachment of PRV-Be, PRV509, and PRV523 to heparinase-treated PK15 (black bars) and Vero (shaded bars) cells. Attachment assays on heparinase-treated cells were performed at 40C as described in Materials and Methods except in the case represented by the hatched bar, which indicates the input PFU of PRV-Be that absorbed to heparinase-treated Vero cells when the inoculum was incubated at 37 instead of 40C. The y axis is the average number of PFU per well, expressed as a percentage of the control values. The strains are indicated along the bottom. The number above each bar is the average number of PFU per well (as a percentage of the control value) of that particular strain that were resistant to PBS washing.

wild-type virus attachment and penetration combined (i.e., absorption). When incubated at 37°C, 85% of the control number of input PFU were absorbed by the heparinasetreated Vero cells (hatched bar, Fig. 6). This rise in infectivity was significant compared with either the 18% determined for stable attachment of PRV-Be at 4°C on heparinase-treated Vero cells (shaded bar, Fig. 6) or the 14% determined for penetration at 37°C on untreated Vero cells (Fig. 4B). Thus, removal of the initial cellular receptor diminished wild-type virus attachment, as expected, but concomitantly relieved the block in wild-type virus penetration of Vero cells.

DISCUSSION Previous work has demonstrated that glycoprotein gIII, the PRV homolog of HSV gC, interacts with cell surface heparan sulfate and functions as the initial viral receptor (17). These results have been obtained in part with virions completely lacking gIII that remain infectious but have been shown to always penetrate cells at markedly reduced rates (15, 36). Thus, it has been concluded that gIII is required for efficient virus infection but is nonessential in cell culture (32). We have extended the genetic studies on gIII to include mutants that have been deleted for major segments of the glycoprotein but still retain an altered form of glll in the virus envelope. Our results contribute to the understanding of initial virus attachment to target cells in two major areas. A functional receptor domain of gIll resides within the amino-terminal one-third of the glycoprotein. Of the strains that contained an altered form of gIII in their envelopes, only PRV523 exhibited an attachment defect at 4°C. This strain, whose giII allele was deleted for codons 25 to 157, infected monolayers efficiently under normal conditions. However, when PBS washes were performed, PRV523 exhibited only residual binding activity to either PK15 or Vero cells. In contrast, strains PRV2 (deletion of codons 158 to 291) and PRV505 (deletion of codons 265 to 411) proved to be

resistant to PBS washing and were therefore as attachment proficient as wild-type virus. It is always difficult to rule out conformational influences on functional domains, but the adjacent and sometimes overlapping nature of the three deletions in PRV523, -2, and -505 strongly argues that a wild-type attachment domain has been deleted in PRV523 and therefore lies in the amino-terminal one-third of gIII. It is important to note that the low level of attachment observed for PRV523 was relatively unaffected by competition with exogenously added heparin or by pretreatment of cell monolayers with heparinase. Thus, the residual binding activity did not appear to be related to wild-type viral attachment to heparan sulfate. Rather, the small numbers of attached PRV523 (and for that matter, PRV509) virions at 4°C probably represent particles that have advanced to a secondary receptor, albeit with poor efficiency. Determining that the residual binding activity of PRV523 virions is unrelated to wild-type attachment is notable because even the truncated form of PRV523-encoded gIll retains clusters of charged amino acids that could serve as heparin-binding domains (HBDs) and, hence, potentially as functional receptor sites. Cardin and Weintraub (2) have offered two consensus sequences for HBDs based on experimental data and the results of computer homology searches. Glycoprotein gIII contains one imperfect and two perfect matches to the consensus sequences in its first one-third and also harbors one perfect and three imperfect potential HBDs in the remaining two-thirds of its polypeptide. If any of these sequences has a role in virus attachment, we can limit our search to one or more of the three amino-terminal sites. Either the remaining four sites in the mutant gIll are not accessible to heparan sulfate or they are somehow excluded from mediating a productive attachment to PK15 or Vero cells. The gC glycoproteins of HSV type 1 (HSV-1) and HSV-2 also contain multiple clusters of charged amino acids that differ from each other and from the potential HBDs of PRV gIII. The differences in the distribution of basic amino acids that are found among the three homologs may indicate that these viruses attach to cells by a similar mechanism but do so by seeking different anionic regions of the very heterogeneous heparan sulfate moieties present on cell surfaces. Glycoprotein gIll-mediated attachment is not required for efficient PRV penetration of all cell types. It has been reported that PRV gIll- and HSV gC- virions are not only attachment deficient but also penetration deficient on all cell types assayed to date (7, 15, 36). This is believed to be an indirect effect resulting from inefficient concentration of virus particles on the cell surface because of failed initial binding to heparan sulfate moieties. We too have found that the attachment-deficient strains PRV509 and PRV523 penetrate PK15 cells slowly, as judged from their extreme sensitivity to acid pH citrate treatment at 1 h postinfection at 37°C. However, on Vero cells, the virions of the same strains penetrated rapidly in the absence of stable attachment, with at least half of the input PFU being resistant to acid pH citrate treatment at 1 h postinfection. One possible explanation of this result is that the virus particles are efficiently endocytosed. We have not assayed for this, but it has been shown previously that the endocytosis of HSV-1 particles by BJ cells constitutively expressing glycoprotein gD did not lead to productive infection (1). Alternatively, the efficient penetration of Vero cells by PRV523 and PRV509 may relate to the two-receptor mechanism that alphaherpesviruses use for entry into cells. Perhaps the mutant virions are able to bypass the requirement for surface concentration that is afforded by heparan

VOL. 67, 1993

ATTACHMENT MUTANTS OF PRV gIIl

sulfate binding because the secondary receptor is more accessible to or has higher affinity for incoming particles on Vero cells than on PK15 cells. In addition to the penetration proficiency of our mutant strains on Vero cells, we found that virions of our wild-type strain, PRV-Be, were apparently blocked for penetration through 1 h postinfection even though they attached to Vero cells efficiently in a heparan sulfate-dependent manner. When Vero cells were first treated with heparinase, the wild-type virus mimicked the mutant strains PRV523 and PRV509, attaching poorly but penetrating rapidly. Therefore, it appears that it is the binding of virus particles to heparan sulfate via wild-type gIII that directly prevents timely penetration, perhaps because the attached particles are misdirected away from the secondary receptor on Vero cells. This would require the assumption that the anionic charge distribution bound by wild-type gIII allows accession to the secondary receptor in PK15 cells but not in Vero cells. However, this seems unlikely, because all of the other attachment-proficient strains were capable of efficient penetration of Vero cells. Alternatively, perhaps wild-type gIII binds Vero (but not PK15) cell surfaces too tightly to allow rapid penetrationthe virus particles simply become too encumbered with heparan sulfate to proceed efficiently to the next step in entry. This high avidity would depend on the level of wild-type gIII, because the gIII signal sequence mutants PRV500 and PRV501 contain reduced amounts of otherwise wild-type gIII in their envelopes yet attach and penetrate efficiently. Lower levels of gIII could lessen avidity simply by reducing the number of nucleation sites for gIII-heparan sulfate interaction or by limiting the amount of gIII available to form multimeric complexes perhaps necessary for avid binding. It should be noted, however, that no evidence exists for homotypic or heterotypic complexes of gIll (33). We have indicated that PRV2 and PRV505 virions may also contain reduced levels of gIII, but these mutants may escape tight binding to heparan sulfate because their gross alterations in gIll indirectly affect avid binding. Heparan sulfate offers virtually limitless combinations of polyanionic charge distribution on the surface of target cells. It is no longer a question of whether heparan sulfate serves as a receptor for certain herpesviruses. It is not clear, however, whether virions and cells interact electrostatically in a nonspecific way or instead must bind through specific alignments of positive charges donated by the virus and negative charges donated by the cell. We suggest that the pertinent basic residues of PRV gIII involved in virus binding lie in the amino-terminal third of the glycoprotein. Moreover, discrete stretches of positively charged amino acids (i.e., HBDs) that could work alone or in concert to mediate a specific electrostatic event that leads to productive virus binding of certain cell types may be identified in this region. This is perhaps required even though the virus contains additional structures capable of interacting with heparinlike molecules. Even so, this binding is not essential for the rapid penetration of all cell types in culture but may have consequences for PRV propagation or virulence in vivo

(16).

ACKNOWLEDGMENTS We thank Kimberly Solomon, Alan Robbins, Mary Whealy, and Lynn Enquist for plasmid pKS1001 and antisera 282 and 284. Oligonucleotides were provided by the Molecular Resource Center, University of Tennessee, Memphis. We also appreciate the critical

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reading of the manuscript prior to its submission by Laraine Powers and Mark Tomilo. This work was supported by grant A128520 from the National Institute of Allergy and Infectious Diseases to P.R. REFERENCES 1. Campadelli-Fiume, G., M. Arsenakis, F. Farabegoli, and B. Roizman. 1988. Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus. J. Virol. 62:159-167. 2. Cardin, A. D., and H. J. R. Weintraub. 1989. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis 9:21-32. 3. 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. 4. Flynn, S., and P. Ryan. Unpublished data. 5. Fuller, A. O., and W. C. Lee. 1992. Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration. J. Virol. 66:50025012. 6. Graham, F. L., and A. S. Van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456-467. 7. Herold, B. C., D. WuDunn, N. Soltys, and P. G. Spear. 1991. Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J. Virol. 65:1090-1098. 8. Highlander, S. L., D. J. Dorney, P. J. Gage, T. C. Holland, W. Cai, S. Person, M. Levine, and J. C. Glorioso. 1989. Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration. J. Virol. 63:730-738. 9. 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. 10. Johnson, D. C., and M. W. Ligas. 1988. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J. Virol. 62:4605-4612. 11. Kjellen, L., and U. Lindahl. 1991. Proteoglycans: structures and interactions. Annu. Rev. Biochem. 60:443-475. 12. Klupp, B. G., N. Visser, and T. C. Mettenleiter. 1992. Identification and characterization of pseudorabies virus glycoprotein H. J. Virol. 66:3048-3055. 13. Lindahl, U., D. S. Feingold, and L. Roden. 1986. Biosynthesis of heparin. Trends Biochem. Sci. 11:221-225. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory,

Cold Spring Harbor, N.Y.

15. Mettenleiter, T. C. 1989. Glycoprotein gIII deletion mutants of pseudorabies virus are impaired in virus entry. Virology 171: 623-625. 16. Mettenleiter, T. C., C. Schreurs, F. Zuckermann, T. Ben-Porat, and A. S. Kaplan. 1988. Role of glycoprotein gIII of pseudorabies virus in virulence. J. Virol. 62:2712-2717. 17. Mettenleiter, T. C., L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat. 1990. Interaction of glycoprotein gIll with a cellular heparinlike substance mediates adsorption of pseudorabies virus. J. Virol. 64:278-286. 18. Neyts, J., R. Snoeck, D. Schols, J. Balzarini, J. D. Esko, A. Vanschepdael, and E. Declercq. 1992. Sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate. Virology 189:48-58. 19. Okazaki, K., T. Matsuzaki, Y. Sugahara, J. Okada, M. Hasebe, Y. Iwamura, M. Ohnishi, T. Kanno, M. Shimizu, E. Honda, and Y. Kono. 1991. BHV-1 adsorption is mediated by the interaction of glycoprotein glll with heparinlike moiety on the cell surface. Virology 181:666-670. 20. Petrovskis, E. A., J. G. Timmins, M. A. Armentrout, C. C.

2654

21.

22.

23.

24. 25.

26.

27.

28.

FLYNN ET AL.

Marchioli, R. J. Yancey, Jr., and L. E. Post. 1986. DNA sequence of the gene for pseudorabies virus gp5O, a glycoprotein without N-linked glycosylation. J. Virol. 59:216-223. Petrovskis, E. A., J. G. Timmins, and L. E. Post. 1986. Use of Agtll to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins. J. Virol. 60:185-193. Robbins, A. K., D. J. Dorney, M. W. Wathen, M. E. Whealy, C. Gold, R. J. Watson, L. E. Holland, S. D. Weed, M. Levine, J. C. Glorioso, and L. W. Enquist. 1987. The pseudorabies virus gll gene is closely related to the gB glycoprotein gene of herpes simplex virus. J. Virol. 61:2691-2701. 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 gIII is not essential for growth in tissue culture. J. Virol. 59:635-645. Ryan, J. P., A. Robbins, M. Whealy, and L. W. Enquist. 1993. Overall signal sequence hydrophobicity determines the in vivo translocation efficiency of a herpesvirus glycoprotein. Virus Genes 7:5-21. Ryan, J. P., M. E. Whealy, A. K. Robbins, and L. W. Enquist. 1987. Analysis of pseudorabies virus glycoprotein glll 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. 1988. Genetic analysis of a herpesvirus glycoprotein signal sequence, p. 299-308. In R. Compans, A. Helenius, and M. B. A. Oldstone (ed.), Cell biology of virus entry, replication and pathogenesis. Alan R. Liss, Inc., New York. Schreurs, C., T. C. Mettenleiter, F. Zuckermann, N. Sugg, and T. Ben-Porat. 1988. Glycoprotein gIII of pseudorabies virus is

J. VIROL. multifunctional. J. Virol. 62:2251-2257. 29. Solomon, K. A., A. K. Robbins, and L. W. Enquist. 1991. Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export. J. Virol. 65:5952-5960. 30. Solomon, K. A., A. K. Robbins, M. E. Whealy, and L. W. Enquist. 1990. The putative cytoplasmic domain of the pseudorabies virus envelope protein gIll, the herpes simplex virus type 1 glycoprotein C homolog, is not required for normal export and localization. J. Virol. 64:3516-3521. 31. Stein, D., and P. Ryan. Unpublished data. 32. Whealy, M. E., A. K. Robbins, and L. W. Enquist. 1988. Pseudorabies virus glycoprotein gIII is required for efficient growth in tissue culture. J. Virol. 62:2512-2515. 33. Whealy, M. E., A. K. Robbins, and L. W. Enquist. 1990. The export pathway of the pseudorabies virus gB homolog gll involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus. J. Virol. 64:19461955. 34. Wittels, M., and P. G. Spear. 1990. Penetration of cells by herpes simplex virus does not require a low-pH-dependent endocytic pathway. Virus Res. 18:271-290. 35. WuDunn, D., and P. G. Spear. 1989. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63:52-58. 36. Zsak, L., T. C. Mettenleiter, N. Sugg, and T. Ben-Porat. 1990. Effect of polylysine on the early stages of infection of wild type pseudorabies virus and of mutants defective in gIll. Virology 179:330-338. 37. Zsak, L., N. Sugg, T. Ben-Porat, A. K. Robbins, M. E. Whealy, and L. W. Enquist. 1991. The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment. J. Virol. 65:4317-4324.