Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.     

Elevated Ca2+(i) transients induced by trimethyltin chloride in HeLa cells: types and levels of response

Humans are exposed to organotins, like trimethyltin (TMT) chloride via air, water and food, and intoxication might result in severe health complications. Toxic effects of organotin compounds are well documented, but possible mechanisms remain unclear and only little information is available how organometallic species interact with calcium controlling mechanisms. Therefore, the aim of this work was to investigate the effects of TMT on calcium homeostasis in HeLa S3 cells. Dynamic changes of cytosolic calcium (Ca2+(i)) were monitored using laser-scanning microscopy and fluo-4 loaded cells. Application of TMT resulted in sustained as well as in transient elevations of Ca2+(i). The number of reacting cells was directly correlated to the concentration of TMT used: with 500 microM TMT all cells reacted, with 50 microM TMT 80% and with 5 microM 74%. The fast Ca2+(i)-transients (spikes), measured in single cells, occurred even with 0.25 microM TMT and varied in size and duration. The sustained increase of Ca2+(i), measured as the average over all cells, was dose dependent with an approximately 8% increase for 5 microM TMT, approximately 12.3% for 50 microM and approximately 145% for 500 microM TMT. Moreover, this effect was partly reversible. A second application resulted in a similar sustained rise of Ca2+(i) compared to the first application of TMT, there was also no difference when no calcium was added to the external solution (151+/-10% compared to 145+/-15%; 500 microM TMT). This rise of Ca2+(i) was highly reduced (<10% increase) when the internal calcium stores were depleted before TMT (500 microM) was applied. Our data suggest that TMT influences Ca2+(i)-homeostasis of HeLa S3 cells, which might be related to its toxicity in this cell line.     

Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains.

Syncytial (syn) mutants of herpes simplex virus cause cell fusion. Many syn mutations map to the syn1 locus, which has been identified with the gK (UL53) gene. In this work, the gK genes of eight syn mutants derived from the KOS strain were sequenced to identify residues and, possibly, domains important for the fusion activity of mutant gK. DNA sequencing showed that six mutants (syn30, syn31, syn32, syn102, syn103, and syn105) had single missense mutations in the gK gene. Two of these, syn31 and syn32, had identical mutations that caused the introduction of a potential site for N-linked glycosylation. syn31 gK was analyzed by in vitro translation and found to utilize the novel glycosylation site. Two other mutants, syn8 and syn33, had three mutations each, resulting in three amino acid substitutions in syn8 and two substitutions in syn33. Of the 10 gK syn mutant sequences known, 8 have mutations in the N-terminal domain of gK, suggesting that this domain, which is likely to be an ectodomain, is important for the function of the protein. The other two mutants, syn30 and syn103, have mutations near the C terminus of gK.     

Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK.

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Herpes simplex virus type 1 gK is required for gB-mediated virus-induced cell fusion, while neither gB and gK nor gB and UL20p function redundantly in virion de-envelopment

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virus-induced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbacDeltagBDeltagK was used to isolate the recombinant viruses gBsyn3DeltagK and gBamb1511DeltagK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3- and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.     

Plasma membrane topology of syncytial domains of herpes simplex virus type 1 glycoprotein K (gK): the UL20 protein enables cell surface localization of gK but not gK-mediated cell-to-cell fusion

Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gK-transfected cells with the gK-null virus DeltagK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.     

Electrically triggered all-or-none Ca(2)+-liberation during action potential in the giant alga Chara.

Electrically triggered action potentials in the giant alga Chara corallina are associated with a transient rise in the concentration of free Ca(2)+ in the cytoplasm (Ca(2)+(cyt)). The present measurements of Ca(2)+(cyt) during membrane excitation show that stimulating pulses of low magnitude (subthreshold pulse) had no perceivable effect on Ca(2)+(cyt). When the strength of a pulse exceeded a narrow threshold (suprathreshold pulse) it evoked the full extent of the Ca(2)+(cyt) elevation. This suggests an all-or-none mechanism for Ca(2)+ mobilization. A transient calcium rise could also be induced by one subthreshold pulse if it was after another subthreshold pulse of the same kind after a suitable interval, i.e., not closer than a few 100 ms and not longer than a few seconds. This dependency of Ca(2)+ mobilization on single and double pulses can be simulated by a model in which a second messenger is produced in a voltage-dependent manner. This second messenger liberates Ca(2)+ from internal stores in an all-or-none manner once a critical concentration (threshold) of the second messenger is exceeded in the cytoplasm. The positive effect of a single suprathreshold pulse and two optimally spaced subthreshold pulses on Ca(2)+ mobilization can be explained on the basis of relative velocity for second messenger production and decomposition as well as the availability of the precursor for the second messenger production. Assuming that inositol-1,4,5,-trisphosphate (IP(3)) is the second messenger in question, the present data provide the major rate constants for IP(3) metabolism.     

Identification and characterization of a novel herpes simplex virus glycoprotein, gK, involved in cell fusion.

Antipeptide sera were used to identify a novel glycoprotein encoded by the UL53 gene of herpes simplex virus type 1 (HSV-1). The UL53 gene product is thought to play a central role in regulating membrane fusion because mutations giving rise to the syncytial phenotype, wherein cells are extensively fused, frequently map to this gene. A single 40-kDa protein, designated gK (the ninth HSV-1 glycoprotein to be described), was detected with antipeptide sera in cells infected with both wild-type and syncytial strains of HSV-1 which were labelled with [35S]methionine and [35S]cysteine or with [3H]glucosamine, and this protein was sensitive to treatment of cells with tunicamycin. With all other HSV glycoproteins studied to date, at least two glycosylated species, often differing substantially in electrophoretic mobility, have been observed in infected cells; thus, gK is unusual in this respect. The 40-kDa gK protein was also immunoprecipitated from cells infected with a recombinant adenovirus vector carrying the UL53 gene. Two glycosylated species of 39 and 41 kDa were produced when UL53 mRNA was translated in vitro in the presence of microsomes, and these proteins differed from gK produced in infected cells not only because they possessed different electrophoretic mobilities but also because they were unable to enter gels after being heated. In addition, a 36-kDa protein was detected in extracts from cells infected with HSV-2 with use of these sera.     

Ion conductance of the Ca(2+)-activated maxi-K+ channel from the embryonic rat brain.

By using single-channel recording techniques, we measured the conductance (gK) of the Ca(2+)-activated Maxi-K+ channel from the embryonic rat brain, and examined its dependence on K+ ions present in equimolar concentrations on both sides of the membrane patch. With ionic strength maintained constant by substitution of N-methyl-D-glucamine for K+, gK has a sigmoidal dependence upon [K+]. This result has been obscured in previous work by variations in ionic strength, which has a marked effect on single-channel conductance, especially in the limit for which this variable approaches zero. The gK versus [K+] relationship is described, theoretically, by a three-barrier, two-binding-site model in which the barrier that an ion must cross to leave the channel is decreased as [K+] is increased.     

On the role of sodium ions in the regulation of the inward-rectifying potassium conductance in cat ventricular myocytes

The conductance of the inward-rectifying K+ current (IK1) in isolated cat ventricular myocytes is decreased by reducing the extracellular Na+ concentration. Using a whole-cell patch-clamp technique, possible mechanisms underlying this Na+ dependence were investigated. These included (a) block of inward K+ current by the Na+ substitute, (b) changes in membrane surface charge associated with removal of extracellular Na+, (c) increases of intracellular Ca2+ due to suppression of Na-Ca exchange, (d) reduction of a Na+-dependent K+ conductance due to a subsequent decrease of intracellular Na+, (e) reduction of IK1 conductance (gK1) associated with reduction of intracellular pH due to suppression of Na-proton exchange. The findings support the hypothesis that the effect of removing Na+ is mediated through a decrease in intracellular pH. These include observations that: (a) reducing internal pH by reducing external pH caused a decrease in gK1, and the conductance changes caused by reducing extracellular pH and removing extracellular Na+ were not additive: (b) the effect of reducing pHo was attenuated by dialyzing with a low pH internal solution; (c) gK1 was reduced by exposure to the Na-proton exchange inhibitor dimethylamiloride, and this effect was absent in the absence of Na+. These findings imply that physiological or pathological processes such as ischemia and metabolic or respiratory acidosis which can produce intracellular acidosis should be expected to affect K+ permeation through the IK1 channel.     

The absence of glycoprotein gL, but not gC or gK, severely impairs pseudorabies virus neuroinvasiveness

Penetration and propagation of herpesviruses in the nervous system require the action of several glycoproteins. To assay for a function of glycoproteins gC, gK, and gL in the neuroinvasiveness of pseudorabies virus (PrV), deletion mutants lacking one of these glycoproteins and corresponding rescuants were inoculated in the nasal cavity of adult mice. We demonstrate that the lack of gL almost prevented the virus from penetrating and propagating in trigeminal, sympathetic, and parasympathetic tracks innervating the nasal cavity, while the lack of gC and gK only slowed the invasion of the nervous system. The conclusion of this and previous studies is that only gB, gD, gH, and gL are indispensable for penetration into neurons, while gB, gH, and gL (and, in some categories of neurons, also gE and gI) are necessary for transneuronal transfer in the mouse model. The deletion of other glycoprotein genes has little effect on PrV neuroinvasiveness although it may affect the dissemination of the virus.     

Small conductance potassium channels cause an activity-dependent spike frequency adaptation and make the transfer function of neurons logarithmic

We made a computational model of a single neuron to study the effect of the small conductance (SK) Ca2+-dependent K+ channel on spike frequency adaptation. The model neuron comprised a Na+ conductance, a Ca2+ conductance, and two Ca2+-independent K+ conductances, as well as a small and a large (BK) Ca2+-activated K+ conductance, a Ca2+ pump, and mechanisms for Ca2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca2+ concentration ([Ca2+]i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca2+]i (. Pflügers Arch. 422:223-232; K?hler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709-1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechner's law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.     

Quantitative description of a calcium induced potassium conductance of the rabbit atrial myocardium by variation of the Ca++ concentration

Action potentials of trabeculae from the left rabbit atrium were studied using a modified sucrose gap method. A mathematical treatment of the action potential time course and the phase plane trajectories gives an approximation of a potassium conductance of gK.GK increases be action potential shows a maximum at 1 s.gK reaches a steady state level after 40 s. The experimental findings are interpreted by means a mathematical model of a Ca++-induced K+-conductance.     

Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

The voltage dependence for outward-going current of the Ca-activated K+ conductance (gK(Ca] of the human red cell membrane has been examined over a wide range of membrane potentials (Vm at constant values of [K+]ex, [K+]c and pHc, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K+ and the corresponding (Vm - EK) values. The basic conductance, defined as the outward-current conductance at (Vm - EK) greater than or equal to 20 mV and [K+]ex greater than or equal to 3 mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen, J. Membrane Biol. 95:121-130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activation gK(Ca) was an apparently linear function of voltage (Vm range -40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of approximately 0.4, assuming chemical equilibrium at Vm = 0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of approximately 5 and chemical equilibrium at the given EK value.     

Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB⁻ genome (PrV-gK^gB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.Herpesvirions are complex structures consisting of a nucleoprotein core, capsid, tegument, and envelope. They comprise at least 30 structural proteins (35). Pseudorabies virus (PrV), a member of the Alphaherpesvirinae, is an economically important animal pathogen, causing Aujeszky’s disease in swine. It is also highly pathogenic for most other mammals except higher primates, including humans (28, 45), and a wide range of cultured cells from different species support productive virus replication, reflecting the wide in vivo host range. Envelope glycoproteins play major roles in the early and late interactions between virion and host cell. They are required for virus entry and participate in release of free virions and viral spread by direct cell-to-cell transmission (27, 37). For PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN, have been characterized (20, 27); these glycoproteins are involved in the attachment of virion to host cell (gC and gD), fusion of viral envelope and cellular cytoplasmic membrane (gB, gD, gH, and gL), spread from infected to noninfected cells (gB, gE, gH, gI, gL, and gM), and egress (gC, gE, and gI) (27, 37). Homologs of these glycoproteins are also present in other alphaherpesviruses (37). The gene coding for a potential 11th PrV glycoprotein, gK, has been described recently (3), but the protein and its function have not been identified.The product of the homologous UL53 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) is gK (13, 32). gK was detected in nuclear membranes and in membranes of the endoplasmic reticulum but was not observed in the plasma membrane (14). Also, it did not appear to be present in purified virion preparations (15). The latter result was surprising since earlier studies identified several mutations in HSV-1 gK resulting in syncytium-inducing phenotypes (7, 14), which indicates participation of gK in membrane fusion events during HSV-1 infection. Moreover, HSV-1 mutants in gK exhibited a delayed entry into noncomplementing cells, which is difficult to reconcile with absence of gK from virions (31). Mutants deficient for gK expression have been isolated and investigated by different groups (16, 17). Mutant F-gKβ carries a lacZ gene insertion in the HSV-1 strain F gK gene, which interrupts the ORF after codon 112 (16). In mutant ΔgK, derived from HSV-1 KOS, almost all of the UL53 gene was deleted (17). Both mutants formed small plaques on Vero cells, and virus yield was reduced to an extent which varied with the different confluencies of the infected cells, cell types, and mutants used for infection. However, both HSV-1 gK mutants showed a defect in efficient translocation of virions from the cytoplasm to the extracellular space, and only a few enveloped virions were present in the extracellular space after infection of Vero cells (16, 17). The authors therefore suggested that HSV-1 gK plays a role in virion transport during egress.Different routes of final envelopment and egress of alphaherpesvirions are discussed. It has been suggested that HSV-1 nucleocapsids acquire their envelope at the inner nuclear membrane and are transported as enveloped particles through the endoplasmic reticulum to the Golgi stacks, where glycoproteins are modified in situ during transport (5, 6, 19, 39), although other potential egress pathways cannot be excluded (4). In contrast, maturation of varicella-zoster virus and PrV involves primary envelopment at the nuclear membrane, followed by release of nucleocapsids into the cytoplasm and secondary envelopment in the trans-Golgi area (10, 12, 43). Final egress of virions appears to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. The possibility of different routes of virion egress is supported by studies of other proteins involved in egress, e.g., the UL20 proteins of HSV-1 and PrV and the PrV UL3.5 protein, which lacks a homolog in the HSV-1 genome (1, 8, 9). In UL20-negative HSV-1, virions accumulated in the perinuclear cisterna of Vero cells (1), while PrV UL20⁻ virions accumulated and were retained in cytoplasmic vesicles (9). PrV UL3.5 is important for budding of nucleocapsids into Golgi-derived vesicles during secondary envelopment (8). Thus, there appear to be profound differences in the egress pathways. Since HSV-1 gK was also implicated in egress, we were interested in identifying the PrV homolog and analyzing its function.     

Binding of HSV-1 Glycoprotein K (gK) to Signal Peptide Peptidase (SPP) Is Required for Virus Infectivity

Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.