Abstract
Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-prolinecysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline 438 induces bending at the end of an alpha-helix, thereby placing cysteine 404 and cysteine 439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine 438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline 438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine 404 to serine. Although PrV expressing gHC 404 S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine 438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59.
Abstract
The role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline 438 in PrV gH to allow the formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions: mutation of this residue resulted in a drastic impairment of membrane fusion kinetics not only in vitro in transient transfection-fusion assays but also during virus infection. Elimination of formation of the disulfide bond yielded the same phenotype in transient assays but had a more drastic effect on virus replication. Thus, our studies add important information to structure-function analyses of herpesvirus gH.
I
n contrast to many other known viral fusion systems which rely on one or two proteins, herpesvirus membrane fusion requires the concerted action of at least three viral glycoproteins (g). In the prototypical alphaherpesvirus, herpes simplex virus 1 (HSV-1), binding of gD to one of its specific host cell receptors induces a conformational change that, in turn, triggers the actual fusion process by signaling to the gH/gL heterodimer and/or gB (1) (2) (3) (4) . gB and gH/gL are essential for membrane fusion during entry as well as for direct viral cell-to-cell spread and are therefore considered the core fusion machinery of herpesviruses. While gB shows typical features of class III fusion proteins, it is not sufficient by itself to induce efficient membrane fusion but requires the presence of gH/gL. The function of this complex, whose structure does not resemble that of any known fusion protein (5-7), still remains elusive (reviewed in reference 8).
I
Herpesvirus gH molecules are type I transmembrane proteins with several N-glycosylation consensus sequences in the ectodomain and a short cytoplasmic tail following the C-terminal membrane anchor. Amino acid sequences are only moderately conserved across the Herpesviridae and are largely limited to several regions within the C-terminal half of the ectodomain (5, 9) . One of the conserved sequence motifs corresponds to amino acids 437 -SPCAVSLRRDL- 447 in gH of the alphaherpesvirus pseudorabies virus (PrV), encompassing a highly conserved serine-proline-cysteine motif, and a second region close to the predicted transmembrane region, 620 -LFPNGTV- 626 , comprising a conserved N-glycosylation site (9) . The preservation of several other cysteine residues throughout the ectodomain already implied similarity of the tertiary structures (9) , which was confirmed by comparison of the crystal structures of the herpes simplex virus 2 (HSV-2) and Epstein-Barr virus (EBV) gH/gL complexes as well as the PrV gH core domain (5) (6) (7) .
I
All known gH homologs form a heterodimeric complex with gL, a small glycoprotein (156 amino acids [aa] in PrV) which depends on gH interaction for membrane association and virion incorporation (10) (11) (12) (13) . While in many herpesviruses gH also requires gL for correct processing and transport, PrV, bovine herpesvirus 4 (BoHV-4), and murine herpesvirus 4 (MuHV-4) gH is also incorporated into virions in the absence of gL (14) (15) (16) . However, in contrast to BoHV-4 and MuHV-4, where gL is not strictly required for fusion, PrV requires gL for entry of free virions (15) , as do all other herpesviruses studied so far. However, direct viral cell-to-cell transmission, which relies on a similar, but not identical, set of proteins, occurs in PrV even in the absence of gL, although at drastically decreased efficiency (15) . This minimal capability for direct viral cell-to-cell spread has been used for serially passaging PrV⌬gL in tissue culture cells, ultimately resulting in a rescuant, PrV⌬gLPass, which efficiently replicated without gL (17) . In this revertant, a hybrid protein is expressed from a fused gene consisting of the receptor binding domain of gD joined in-frame to an N-terminally truncated gH core fragment that lacks the putative gL binding domain (Fig. 1 ). This gDH hybrid protein is sufficient to induce membrane fusion in combination with gB and complements the defect of mutants simultaneously lacking gD, gH, and gL (17, 18) , indicating that membrane fusion can be accomplished without gL and with a minimal set of two herpesviral proteins.
I
From the crystal structures of the ectodomains of HSV-2 and EBV gH/gL (6, 7), as well as the core fragment of PrV gH, which was derived from the gDH hybrid protein (5), four distinct domains could be defined. While domain I, which is involved in binding gL, is missing in the PrV gH structure (Fig. 1) , domains II to IV are largely superimposable among the three proteins. Domain II contains two conserved structures, an antiparallel betasheet designated the "fence" and an elongated antiparallel 3-helix bundle which shows structural similarities to a domain of cellular syntaxins and has accordingly been designated the "syntaxin-like bundle" (SLB) (5) (Fig. 2) . Domain III consists mainly of alphahelices and harbors one of the conserved sequence motifs, including residues serine 437 , proline 438 , and cysteine 439 (numbering for PrV gH), with proline and cysteine invariably present in all herpesvirus gH homologs. Proline 438 induces bending at the end of an alpha-helix, so that cysteine 439 is brought into proximity to cysteine 404 and is able to form a strictly conserved disulfide bond with cysteine 404 (Fig. 2 ) (5). The membrane-proximal domain IV, which shows the highest conservation, is composed of two fourstranded beta-sheets connected by the so-called "flap" region. This hydrophilic flap, which is followed by a conserved N-glycosylation site, covers a hydrophobic patch, which was shown to play an important role during fusion. It is speculated that movement of the flap, which masks the underlying hydrophobic patch, promotes membrane fusion (5, 19) .
I
Recently, the complete genome sequence of PrV strain Bartha (PrV-Ba), an attenuated strain widely used as a vaccine against PrV infection in pigs, was deciphered and compared to wild-type strains PrV-Kaplan (PrV-Ka) and PrV-Becker (20) . While the attenuating mutations in PrV-Ba were mapped earlier by marker rescue experiments (21) (22) (23) (24) and were shown to be a result of a large deletion within the unique short region comprising genes encoding gI, gE, pUS9, and pUS2, as well as subtle amino acid changes in pUL21 and gC, comparison of the deduced amino acid sequences revealed a total of 46 open reading frames with nonsilent mutations (20) . Among these mutations, an alteration in gH
I
The gH open reading frames are shown as rectangles. The core fragment of gH present in the gDH hybrid protein, which had been used for crystallization, is presented above. The domains are labeled with roman numbers with boundaries shown. The black diamond indicates the location of the SPC motif. The shaded boxes left and right indicate residues that were not resolved in the structure. The predicted signal sequence (SS; aa 1 to 30) is highlighted in black; the transmembrane domain (TM; aa 647 to 663) is indicated in gray. The location of the PmlI restriction site used for cloning is shown. Amino acid differences between PrV-Ba gH and PrV-Ka gH are given in one-letter code with the corresponding position.
I
within the highly conserved sequence motif 437 serine-proline-cysteine 439 was identified. Interestingly, PrV-Ba gH contains a serine instead of the helix-bending proline (20) .
I
Since proline 438 is strictly conserved throughout other herpesvirus gH homologs, as is the disulfide bridge between cysteine 404 and cysteine 439 (5, 9), we were interested in analyzing the effect of this alteration on gH function. To this end, we generated PrV gH variants expressing either serine or proline at position 438 as well as a serine mutant at position 404 and analyzed them in transient transfection-fusion assays and, after rescue of the respective recombinant viruses in PrV-Ka, in one-step growth, penetration kinetics, and cell-to-cell transmission assays.
MATERIALS AND METHODS

Cells and viruses.
Rabbit kidney cells (RK13) and RK13-gHgL cells (25) were grown in Dulbecco's modified Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum at 37°C in the presence of 5% CO 2 . Recombinants used in this study were derived from pPrV⌬gGG, comprising the PrV-Ka genomic DNA cloned as a bacterial artificial chromosome (BAC) with the mini-F plasmid sequence and a green fluorescent protein (GFP) expression cassette at the nonessential gG gene locus (19) . In pPrV-⌬gHABF (for simplicity, here designated pPrV-⌬gH), gH codons from 51 to the end were deleted (S. Böhm, E. Eckroth, M. Backovic, B. G. Klupp, F. A. Rey, T. C. Mettenleiter, and W. Fuchs, unpublished data). pPrV-gH Ka was generated by reinsertion of the wild-type Kaplan gH (gH Ka ) together with a downstream-located kanamycin resistance gene (kan) for selection in Escherichia coli and was used here as the wild-type control. For comparison, a BAC clone (here designated pPrVBa) derived from PrV-Bartha (pPrV-Ba⌬gGG [26] ), generated after deletion and reinsertion of the Bartha gH gene (gH Ba ), including the downstream kan, was used. The gH deletion mutants were grown in RK13-gHgL cells, while all the other recombinants were propagated on RK13 cells.
Cells and viruses.
Expression plasmids and gH mutagenesis. Plasmids pcDNA-gB, pcDNA-gH, and pcDNA-gL express the full-length gB, gH, and gL of PrV-Ka, respectively, under the control of the human cytomegalovirus immediate early promoter/enhancer complex (18) . pcDNA-gD expressing PrV-Ka gD was generated after PCR amplification using primers gD-fw and gD-rv (Table 1) and PrV-Ka genomic DNA as a template. The 1.2-kb PCR product was cloned into pcDNA3 (Invitrogen) after cleavage with EcoRI using the corresponding restriction sites added by the primers. pcDNA-gH Ba was generated using primers DH-3 and DHrev1 (Table 1 ) on genomic PrV-Ba DNA (27) . The 2.1-kb PCR product was cloned into pcDNA3 using the EcoRI and XbaI restriction enzyme sites provided by the primers. Correct amplification and insertion were verified by sequencing and indirect immunofluorescence assay after transfection (data not shown). For sequencing, a BigDye Terminator cycle sequencing kit (Applied Biosystems) was used, and products were run on an ABI 3130 genetic analyzer (Applied Biosystems).
Cells and viruses.
To introduce mutations into the gH sequence, a QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) was used as described recently (28) . Specific primers (Table 1) derived from the genomic sequence of PrV-Ka (GenBank accession no. JQ809328 [29] ) or PrV-Ba (GenBank accession no. JF797217 [20] ) were applied to generate pcDNAgH Ka 5 RK13 cells per well in 24-well culture dishes were seeded the day before transfection. Cells were then transfected with 125 ng of each plasmid expressing one of the gH variants, full-length gB, gD, and gL, as well as pEGFP-N1 (Clontech) as a marker in 50 l of serum-free MEM using 1 l polyethylenimine (PEI). After incubation for 15 min at room temperature, the mixture was added to the cells. pcDNA3 was used as a negative control, substituting for the gH expression plasmid in the transfection assays. Forty-eight hours posttransfection, cells were washed with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde for 20 min, and washed twice with PBS. To quantify the fusion activity, we evaluated 10 randomly selected fields of view in each well by counting the number of green-fluorescing syncytia and measuring the polykaryocyte area using a Nikon Eclipse Ti-S fluorescence microscope and Nikon NIS-Elements imaging software (Nikon). Syncytia were defined as cells with three or more nuclei. Total fusion activity was calculated by multiplication of the number of syncytia by the mean value of the syncytium area in each assay. The experiment was repeated three times, and the average values (values for assays with pcDNA-gH Ka set as 100%) of the results with the corresponding standard deviations are given.
Cells and viruses.
Generation of gH recombinants. The transfer plasmids used for reinsertion of authentic, heterologous, or mutated gH genes into PrV-Ka were based on the expression plasmid pcDNA-gHKDE (Böhm et al., unpublished), comprising the full-length gH Ka followed by kan and a DrdI restriction site. A 1.7-kbp KpnI-BamHI fragment covering most of the gH gene (corresponding to codons 88 to 662) was replaced with the corresponding fragments of pcDNA-gH Ba , pcDNA-gH Ka P 438 S, and pcDNAgH Ka C 404 S to generate pPrV-gH Ba D 59 V, pPrV-gH Ka P 438 S, and pPrVgH Ka C 404 S. The transfer plasmid for pPrV-gH Ka V 59 D was generated after substitution of a 0.5-kbp SanDI-BsrGI fragment (comprising codons 42 to 216) in pcDNA-gHKDE by the corresponding fragment from pcDNAgH Ba . The triplet encoding serine 438 in gH Ba was mutated to a proline codon by site-directed mutagenesis with primers S 438 P-fw and S 438 P-rv (Table 1) , resulting in the transfer plasmid for pPrV-gH Ba S 438 P. By recloning of the 1.7-kb KpnI/BamHI fragment from this plasmid into pcDNA-gHKDE, the transfer plasmid for pPrV-gH Ba D 59 V/S 438 P was obtained. All plasmids were digested with DrdI, and the 3.5-kbp fragments containing the complete gH gene followed by kan were isolated and subsequently used for Red-mediated mutagenesis of pPrV-⌬gH (Böhm et al., unpublished). PrV recombinants were characterized by restriction analyses and Southern blot hybridization of genomic DNA. Correct mutagenesis was verified by PCR amplification and sequencing of the complete gH open reading frame (data not shown).
Cells and viruses.
Western blotting. For immunoblotting, RK13 cells were infected at a multiplicity of infection (MOI) of 3. Twenty-four hours after infection, cells and supernatants were harvested, pelleted by centrifugation, and washed twice with PBS. The pellet was lysed in sodium dodecyl sulfate (SDS) sample buffer, and the proteins were separated in SDS-10% polyacrylamide gels. After blotting onto nitrocellulose and blocking with 5% skimmed milk, membranes were incubated with rabbit sera specific for gH (17) , capsid triplex component pUL38 (unpublished data), or a monoclonal mouse anti-gE antibody (17) . Bound antibodies were detected by incubation with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Dianova). Blots were analyzed using an imager (VersaDoc; Bio-Rad) after incubation with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).
Cells and viruses.
One-step growth kinetics. For analysis of replication properties, RK13 cells were infected at an MOI of 3 and incubated on ice for 1 h. Then the inoculum was replaced by prewarmed medium and the cells were further incubated at 37°C. After 1 h of incubation at 37°C, nonpenetrated extracellular virus was inactivated by low-pH treatment (30) . The cells were harvested at different times thereafter by scraping into the supernatant and were stored at Ϫ80°C. After thawing, the supernatant was titrated on RK13 cells. This assay was repeated two times, and mean values with the corresponding standard deviations were calculated.
Cells and viruses.
Plaque assays. To determine the plaque size, RK13 cells grown in 12-well culture dishes were infected with the different recombinants with approximately 100 PFU under plaque assay conditions and fixed with 3% paraformaldehyde 2 days postinfection (p.i.). The fluorescent area of 30 plaques was measured microscopically with a Nikon Eclipse Ti-S fluorescence microscope using Nikon NIS-Elements imaging software and was compared to the area measured for pPrV-gH Ka , which was set as 100%. Mean values with standard deviations from three independent experiments are shown.
Cells and viruses.
Penetration kinetics. The kinetics of penetration of the different viruses was analyzed in RK13 cells. Cells grown in 12-well culture dishes were infected on ice with approximately 150 PFU of the different recombinants. After 1 h, the inoculum was replaced by prewarmed medium and the cells were incubated at 37°C; after 0, 5, 10, 20, and 40 min, the remaining extracellular virus was inactivated by low-pH treatment in one well, while a parallel well was used as a 100% penetration control and washed with PBS only. Two days p.i., cells were fixed and stained with crystal violet, and plaques were counted. To calculate the penetration rate, we compared the number of PFU surviving the low-pH inactivation to the number of plaques formed in the control. Mean values with standard deviations from three independent assays are given.
RESULTS

Sequencing of PrV-Bartha gH.
Since the published genome sequences of PrV-Ka generated by Szpara et al. (20) and our laboratory (29) differ at several positions in the coding regions, we first verified whether the Bartha strain used by us (Bartha K61) (27) comprises the same mutations in the gH open reading frame as those previously reported (20) . To this end, we amplified the complete gH gene of PrV-Ba genomic DNA and sequenced it after cloning. Comparison of the deduced amino acid sequences revealed changes in PrV-Ba gH identical to those previously reported (20) , namely, V 59 D, P 438 S, EE 481,482 G, and I 539 L (Fig. 1) , thus verifying the proline-to-serine mutation at position 438 within the highly conserved sequence motif (5) . Figure 3 shows the mean values for three independent assays with the corresponding standard deviations. Cotransfection of the plasmids expressing the native PrV-Ka glycoproteins resulted in approximately 60 syncytia in the 10 randomly selected fields of view, which was set as 100%. Substitution of proline 438 with serine (gH Ka P 438 S) led to a ca. 90% reduction of fusion activity resulting in activity similar to that observed in assays with gH Ba and just slightly higher than in assays without any gH expression plasmid (pcDNA3), showing that proline at position 438 plays an important role for gH function during membrane fusion in these transient assays.
Sequencing of PrV-Bartha gH.
Bending of alpha-helix 11 in gH induced by proline 438 was suggested to be important for formation of a highly conserved disulfide bond connecting cysteine 404 and cysteine 439 in PrV gH ( Fig. 2) (5) . To substantiate this assumption, we generated a Ka-gH expression plasmid with cysteine 404 mutated to serine (gH Ka C 404 S). In transient-fusion assays, gH Ka C 404 S yielded reduced fusion activity similar to that of gH Ka P 438 S (Fig. 3) , suggesting that proline 438 is indeed involved in mediating bridging between cysteine 404 and cysteine 439 . Restoring proline 438 in gH Ba (gH Ba S 438 P) resulted in slightly enhanced fusion activity, which, however, did not reach the levels of fusion obtained with gH Ka (Fig. 3) . Thus, to test which other mutation(s) present in gH Ba may influence its fusion activity, we altered other amino acids which differed between PrV-gH Ba and PrV-gH Ka (Fig. 1) ture gH could be demonstrated, and no gH-specific signal was detected in pPrV-⌬gH-infected cells. It is notable that the mutations did not introduce an additional N-linked glycosylation site. The capsid protein pUL38, which served as a loading control, was present in similar amounts in all infected cell lysates, while gE was absent from the Bartha-derived recombinant pPrV-Ba.
Sequencing of PrV-Bartha gH.
The proline 438 serine substitution in PrV gH has no influence on final viral titers. To test for viral replication, RK13 cells were infected at an MOI of 3 with the different viruses. Cells and supernatants were harvested 0, 4, 8, 12, 24, and 32 h after infection, and titers were determined on RK13 cells. The mean values from two independent experiments are shown in Fig. 5 . While the gH deletion mutant pPrV-⌬gH does not productively replicate on RK13 cells, titers of viruses expressing the different gH variants were comparable, demonstrating that the proline 438 serine substitution has no effect on production of infectious progeny. pPrVgH Ka C 404 S proved to be replication competent, but titers were approximately 10-fold lower, indicating that a complete block of formation of the disulfide bond has a greater effect than impairment by mutation of the neighboring proline.
Sequencing of PrV-Bartha gH.
The proline 438 serine substitution in PrV gH affects direct viral cell-to-cell transmission. Since gH is essential for direct viral cell-to-cell spread, plaque areas were measured 2 days after infection. PrV-Ba was already known to form small plaques, primarily due to a cells were cotransfected with plasmids coding for gH Ka , gH Ba , or the respective gH mutants and plasmids encoding the full-length forms of gB, gD, and gL, as well as enhanced green fluorescent protein (EGFP) as a marker. The empty vector pcDNA3 served as a negative control, substituting for the gH expression plasmid. Total fusion activity (number of syncytia ϫ syncytium area) in assays containing gH Ka was set as 100%, and the corresponding standard deviations for three independent assays were calculated. large deletion in the unique short region comprising, among others, the genes encoding gI and gE (22) , which is similar for the BACderived mutant pPrV-Ba (Fig. 6) . Expression of gH Ba by PrV-Ka also resulted in a small-plaque phenotype. This was also found for all mutants expressing gH variants with serine 438 . In contrast, recombinants with proline 438 formed plaques which were comparable to those of pPrV-gH (Fig. 6B) . 
The gH proline
438 serine substitution delays penetration kinetics. Penetration kinetics was analyzed to assess the influence of the proline 438 serine substitution on the rate of fusion during entry. While pPrV-gH Ka entered cells extremely fast, with 80% of input virus protected from low-pH treatment after approximately 5 min, pPrV-gH Ba reached this level only after approximately 20 min. The penetration kinetics of pPrV-gH Ka P 438 S was as slow as that of pPrV-gH Ba , indicating a significant influence of the proline 438 serine substitution on fusion kinetics during virus entry (Fig. 7) . In contrast, the introduction of proline 438 into gH Ba restored penetration kinetics to rates comparable to those for pPrVgH Ka (Fig. 7) . Penetration of pPrV-gH Ka C 404 S was the slowest, but the virus was still able to enter cells and to initiate productive infection.
DISCUSSION
The gH/gL complex is an essential component of the herpesviral fusion machinery, but its role is not well understood. Although an active role in membrane fusion has been suggested for gH (31) (32) (33) , analysis of the available crystal structures showed no signatures of known fusion proteins (5-7). More recent data indicate that the gH/gL complex plays a regulatory role, mediating the timely and efficient transition of gB into its fusion-active state (6). However, how this is accomplished remains unknown.
DISCUSSION
Amino acid sequence conservation is only moderate between the different herpesvirus gH molecules. However, the "SPC" motif located at positions 437 to 439 in PrV gH, with proline and cysteine invariably present in gH homologs of all herpesvirus subfamilies, was thought to be indispensable (5) . It was, therefore, surprising that PrV strain Bartha, an attenuated vaccine strain, carries a serine at position 438 (20) . To test the influence of this unexpected alteration on gH function, we mutated proline 438 to serine in PrV-Ka gH and changed serine 438 to proline in PrV-Ba gH. In transient transfection-fusion assays, a drastic impairment in syncytium formation was evident when RK13 cells were cotransfected with expression plasmids for full-length gB, gD, and gL and plasmids expressing gH with the serine 438 substitution. This indicates that proline 438 plays a major role in supporting efficient cellcell fusion. Since it was speculated that proline 438 is necessary for the bending of alpha-helix 11 as a prerequisite for cysteine 439 to come into the vicinity of and form a disulfide bridge with the conserved cysteine 404 ( Fig. 2 ) (5), we tested whether the replacement of cysteine 404 with serine resulted in the same effect. Fusion assays with gH (34, 35) .
DISCUSSION
The proline 438 serine substitution also affected gH maturation, as demonstrated by the increased presence of immature gH (Fig.  4) . It can be speculated that the presence of serine instead of proline in alpha-helix 11 (5) influences the folding of gH and that incorrectly folded gH molecules are retained in the endoplasmic reticulum (ER) and are not efficiently transported through the Golgi apparatus. Alternatively, improper folding may impair access to glycosylation sites during the transit of gH through the secretory pathway. Nevertheless, PrV-Ba gH, which contains this substitution, is functionally active to support viral replication, as is gH Ka P 438 S, at least in cell culture. In cells expressing gH Ka C 404 S, only immature gH was detected, in line with the importance of this disulfide bond for gH function in HSV and VZV.
DISCUSSION
Whether the proline 438 serine substitution affects virulence in vivo remains to be determined. However, it is important to note that full restoration of the virulence of PrV-Ba was achieved after repair of the deletion in the unique short region and point mutations in pUL21 (21, 23, 36 Ka instead of valine drastically reduced fusogenicity, indicating that this amino acid position is also important for gH function. Since domain I, which also contains the gL binding site, was not part of the gH construct that was crystallized (Fig. 2) , it is unclear whether this mutation might affect domain III directly or whether it influences fusogenicity by affecting gL binding. Unfortunately, the region encompassing V59 cannot be aligned with the structure of the other gH proteins. To analyze the influence of the proline/serine variation during virus infection, we generated PrV-Ka recombinants expressing the different gH molecules. Despite the obvious impairment of syncytium formation in the transfection-fusion assays, the PrV recombinants expressing gH with the serine substitution at position 438 replicated to titers comparable to those of pPrV-gH Ka and pPrV-gH Ba , indicating that the mutation has no effect on virus production after high-MOI infection, which is in line with the notion that once inside the cell, gH is not required for continuing viral replication (25) . In contrast, pPrV-gH Ka C 404 S replicated to ca. 10-fold-reduced viral titers. Although this represents a significant defect, it clearly shows that, in contrast to HSV and VZV, PrV does not require the disulfide bond involving C 404 for gH function. Our data suggest that the P 438 S mutation may impair protein folding such that only a fraction of the molecules folds correctly by a spontaneous tilt of alpha-helix 11, is able to leave the ER, and is properly processed. In contrast, another fraction is retained in the ER and remains in an immature state, as indicated by our protein analyses. Since correctly folded and properly processed gH will be incorporated into progeny virions, infectious particles will form but may contain less gH than the wild type. Maturation of gH is even more impaired by the complete inactivation of the disulfide bond in the C 404 S mutant, which correlates with the more severe phenotypic defects. However, even in the absence of the disulfide bond, PrV gH retains a residual capacity to function during entry.
DISCUSSION
Direct viral cell-to-cell spread as measured by plaque size was significantly reduced in RK13 cells infected with the serine 438 variants, but plaque size could be restored to wild-type levels by serine 438 proline reversions. The strongest phenotype is shown by pPrV-gH Ka C 404 S, which produces only tiny foci of infected cells, yet there is noticeable viral spread compared to infection of only single cells with the gH deletion mutant.
DISCUSSION
The molecular mechanism of direct cell-to-cell transmission of herpesviruses has remained unknown. While it relies on the same set of proteins as fusion of free virions in many herpesviruses, PrV can spread via direct cell-cell transmission in the absence of the receptor binding gD (37, 38) and also, partly, in the absence of gL (15) , while gB and gH are absolutely required. This demonstrates that both processes depend on similar but not identical mechanisms. In addition, other proteins such as the gE/gI complex facilitate the direct transit of infectivity from infected to neighboring uninfected cells. It was suggested that gE is targeted to cell junctions, thereby helping virus spread (39) . In consequence, restoration of the deleted unique short region to PrV-Ba resulted in significantly larger plaque sizes (36) . In our experiments, plaque size could also be increased by just altering serine 438 in PrV-Ba gH expressed in a PrV-Ka background. We are currently analyzing whether in a PrV-Ba background in the presence of the deletion in the unique short region, eliminating expression of gE and gI, alteration of these amino acids in gH has a similar effect.
DISCUSSION
Penetration kinetics showed that the velocity of virus entry is delayed in viruses expressing serine 438 instead of proline. While 80% of pPrV-gH Ka entered cells within approximately 5 min after temperature shift, all mutants expressing gH with serine 438 required longer to reach the same value, indicating that either the fusion at the plasma membrane is less efficient or uptake might take place by a different mechanism, e.g., endocytosis. Thus, the transient transfection-fusion assay, which shows a distinct impairment of gH activity by the proline 438 serine alteration, correlates with the results from penetration kinetics and plaque size determination, but not one-step replication after high MOI infection and final virus titers. Penetration of pPrV-gH Ka C 404 S is slowest, with only ca. 50% of the virus protected from acid inactivation after 40 min. Thus, inability to form this disulfide bond, which is essential in HSV and VZV, strongly impairs gH function in PrV but does not disable it completely. Therefore, despite the significant effect of the C 404 S mutation on PrV gH maturation as well as on viral cell-cell spread, penetration, and one-step replication, the mutated gH is still able to function, although at drastically lower efficiency. This unexpected result demonstrates that it is imperative to perform multiple assays with multiple viruses to unravel the functional consequences of specific alterations within this conserved protein.
DISCUSSION
In summary, alteration of the conserved proline at position 438 of PrV gH to serine influences the fusion activity of the protein in transient assays as well as cell-cell spread and penetration kinetics during infection. It also affects gH maturation. All these effects are mimicked, but increased, by abolition of the disulfide bond involving cysteine 404 . Thus, our data support the assumption that proline 438 assists in, but is not essential for, formation of the cysteine 404 -cysteine 439 disulfide bond by bending alpha-helix 11 and that formation of this disulfide bond is impaired but not eliminated by the amino acid exchange. Therefore, PrV-Ba retains replication competence despite the presence of the proline 438 serine mutation, which is a prime prerequisite for its use as a safe and efficacious PrV vaccine.

{'paper_id': '26001687', 'title': 'The highly conserved proline at position 438 in pseudorabies virus gH is important for regulation of membrane fusion.', 's2_url': 'https://api.semanticscholar.org/CorpusID:26001687', 'source': 'S2ORC'}