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J Virol. Aug 2008; 82(16): 8094–8104.
Published online Jun 4, 2008. doi:  10.1128/JVI.00874-08
PMCID: PMC2519571
Effects of Lamin A/C, Lamin B1, and Viral US3 Kinase Activity on Viral Infectivity, Virion Egress, and the Targeting of Herpes Simplex Virus UL34-Encoded Protein to the Inner Nuclear Membrane [down-pointing small open triangle]
Fan Mou, Elizabeth G. Wills, Richard Park, and Joel D. Baines*
Department of Microbiology and Immunology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
*Corresponding author. Mailing address: C5169 Vet Med Center, Cornell University, Ithaca NY 14853. Phone: (607) 253-3391. Fax: (607) 253-3384. E-mail: jdb11/at/cornell.edu
Received April 24, 2008; Accepted May 28, 2008.
Previous results indicated that the UL34 protein (pUL34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pUL34 and virions requires the US3-encoded kinase that phosphorylates UL34 and lamin A/C. Moreover, pUL34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pUL34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pUL34 targeting to the inner nuclear membrane (INM), the localization of pUL34 was determined in LmnA−/− and LmnB1−/− mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of US3 kinase activity. While pUL34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a US3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pUL34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pUL34 protein in cells infected with a US3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that US3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pUL34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins.
Herpesvirus nucleocapsids are assembled in the nucleus and become enveloped initially at the inner nuclear membrane (INM) in a reaction termed primary envelopment (1). The herpes simplex virus UL31 and UL34 proteins (pUL31 and pUL34, respectively) and their orthologs in other herpesviruses are targeted to the INM and are required for the primary envelopment reaction in many cell lines (8, 19, 27, 28, 31). Upon infection of restrictive cells such as Vero and Hep2 with herpes simplex virus (HSV) deletion mutants lacking UL31 or UL34, nucleocapsids remain in the nucleoplasm and infectious titers are reduced at least 1,000-fold (3, 18, 29).
The UL34 protein is a type II integral membrane protein of 30,000 Mr, with a predicted 250-amino-acid (aa) nucleoplasmic domain, a 23-aa transmembrane domain, and 5 aa in the perinuclear space (21, 32). The UL31 protein is a largely hydrophobic phosphoprotein of 32,000 Mr that requires interaction with pUL34 for targeting to the nucleoplasmic face of the INM (21, 27, 40). While pUL31 and pUL34 are incorporated into nascent virions, they are undetectable in extracellular virions, suggesting they are lost from the nascent virion when its envelope fuses with the outer nuclear membrane (7, 28). This observation is consistent with the most prominent model of virion egress in which the de-enveloped nucleocapsid subsequently gains a new envelope by budding into cytoplasmic membranes (35). In the absence of the activity of a serine kinase encoded by alphaherpesvirus genes, such as HSV-1 US3, or upon deletion of the HSV genes encoding glycoproteins B and H (gB and gH, respectively), virions accumulate aberrantly in the perinuclear space, suggesting that US3, gB, and gH promote the fusion of the nascent virion envelope with the outer nuclear membrane (5, 15, 28, 30, 39). Interestingly, lamin A, pUL31, and pUL34 are phosphorylated by the US3 kinase, and the kinase activity is required for smooth localization of the UL31/UL34 complex throughout the nuclear rim of infected cells (12, 23, 25, 30). Otherwise, the UL31/UL34 complex accumulates aberrantly in discrete foci located at the nuclear rim. At least some of these foci are adjacent to areas of the perinuclear space in which virions accumulate.
How the pUL31/pUL34 complex is targeted to budding sites at the INM is an important but unresolved question. Host proteins are sufficient for targeting the complex to the INM because transiently expressed pUL31 and pUL34 are cotargeted to the nuclear rim in the absence of other viral proteins (27, 40). A current model proposes that most INM-destined integral membrane proteins in the endoplasmic reticulum use a subset of cytosolic nuclear localization signals to target them past the nuclear pore membrane to the INM via a karyopherin β1-driven pathway (13, 20). The proteins then become anchored to the INM by interaction with relatively immobile elements of the nuclear lamina (4). The nuclear lamina comprises a complex network of proteins that line the entire nucleoplasmic surface of the INM, structurally support the nucleus, and interact with chromatin (9). Intermediate filaments made of proteins called lamins comprise the major structural component of the nuclear lamina (6, 22). Lamins consist of two types, designated A and B. An alternatively spliced version of lamin A produces lamin type C that has a unique carboxyl terminus (6). Lamin B is expressed from two homologous genes, which encode lamin B1 and lamin B2 (10, 11). We previously showed that both pUL31 and pUL34 interact with lamin A produced in rabbit reticulocyte lysates, suggesting that this interaction might play a role in anchoring pUL31/pUL34 to the INM (26).
The main goal in initiating the current work was to test whether lamins are required for targeting of the pUL31/pUL34 complex to the inner nuclear membrane.
Cells and viruses.
Wild-type virus HSV-1(F) and a mutant HSV-1(F), containing a lysine-to-alanine mutation in US3 codon 220 (K220A), have been described and were obtained from B. Roizman and R. J. Roller, respectively. The viruses were grown and titers were determined on Vero cells as described previously.
Hep2 cell lines engineered to express green fluorescent protein (GFP) fused to the N terminus of lamin A/C have been described previously (23). An immortalized lamin A/C knockout mouse embryonic fibroblast (MEF) cell line lacking the gene encoding lamin A/C (LmnA−/−) was a gift from Colin Stewart, NIH (37). To enhance cell adherence, LmnA/ cells were propagated on cultureware pretreated with poly-l-lysine before seeding. Control MEFs (3T3) were obtained from Robert Weiss (Cornell University). This treatment precluded adherence of lamin B1 knockout MEFs (LmnB1/), and corresponding wild-type MEFs (LmnB1+/+) were a generous gift from Karen Reue and have been described elsewhere (38). Pretreatment with poly-l-lysine precluded adherence of these cells and was not used in their propagation. All MEFs were grown in growth medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics), except that the LmnB1/ MEFs and their control wild-type cells were supplemented with additional nonessential amino acids (Gibco) and Glutamax (Gibco).
Antibodies and immunofluorescence.
Polyclonal chicken antibody against pUL34 was a kind gift of Richard Roller and has been described elsewhere (29). Purified polyclonal chicken immunoglobulin Y (IgY) directed against lamin A/C was made in our laboratory and has been published previously (26). Mouse monoclonal antibody directed against lamin A/C and goat polyclonal IgG directed against lamin B were purchased from Santa Cruz Biotechnology (catalog numbers sc-7292 and sc-6216, respectively).
For immunofluorescence, MEFs grown on glass coverslips were infected with 5.0 PFU of wild-type HSV-1(F) or US3(K220A) HSV-1 per cell. Thirteen hours later, cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, quenched with 50 mM NH4Cl for 15 min, and then permeabilized with 0.1% Triton X-100. Nonspecific immunoreactivity was blocked by reaction with 10% human serum in PBS for 1 hour at room temperature and another 1 hour with 10% BlockHen II (Aves Lab). The cells were then reacted with anti-pUL34 chicken IgY diluted 1:400 and goat polyclonal anti-lamin B antibody diluted 1:100 in PBS plus 1% bovine serum albumin for 1 h at room temperature as needed. Bound primary antibodies were recognized by Texas Red- or fluorescein isothiocyanate-conjugated anti-immunoglobulins (Jackson ImmunoResearch).
One-step viral growth curves.
LmnA/ and 3T3 MEFs grown in 12-well plates pretreated with poly-l-lysine were infected with 5 PFU of HSV-1(F) or Us3(K220A) HSV-1 per cell. After 1 h of incubation at 37°C, residual surface infectivity was inactivated with a low-pH wash (40 mM citric acid [pH 3.0], 10 mM KCl, 135 mM NaCl). A similar approach was used for the lamin B knockout cells and 3T3 control MEFS infected in parallel, except that cultureware was not pretreated. At the indicated time points, the cultures were frozen and thawed to lyse the cells, and infectious virus was titrated by plaque assay on Vero cells. Mean values of two independent experiments and corresponding standard deviations were calculated and plotted.
GST pull-down assay and mass spectrometry.
Approximately 4 × 108 Hep2 cells were infected with 5 PFU/cell of wild-type HSV-1(F). At 16 h postinfection the cells were lysed in 30 ml modified radioimmunoprecipitation assay buffer {50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM EDTA} containing 1× Complete protease inhibitor cocktail (Roche) and phosphatase inhibitor (10 mM NaF, 10 mM Na3VO4) with gentle rocking for 2 h at 4°C. The lysate was clarified by centrifugation at 10,000 × g for 20 min at 4°C, and the supernatant was reacted with glutathione-Sepharose beads (GE) at 4°C. A glutathione S-transferase (GST)-pUL34 fusion protein was affinity purified from lysates of Escherichia coli using glutathione-Sepharose beads as described previously (26). The fusion protein was then covalently cross-linked to the Sepharose beads by reaction with 5 mM bis(sulfosuccinimidyl)suberate (Pierce) according to the manufacturer's protocol. GST identically cross-linked to Sepharose beads served as a control. After overnight incubation of the precleared HSV-infected cell lysate with the protein-Sepharose beads bearing GST or GST-pUL34 at 4°C, the beads were washed two times with ice-cold modified radioimmunoprecipitation assay buffer and then three times with 0.5% Tween 20-PBS. The bound proteins were eluted by boiling for 10 min in SDS-polyacrylamide gel electrophoresis sample buffer (10 mM Tris-HCl [pH 8.0], 10 mM β-mercaptoethanol, 20% glycerol, 5% SDS, trace amounts of bromophenol blue). The eluted proteins were then separated electrophoretically on a 10% polyacrylamide gel (SDS-polyacrylamide gel electrophoresis) and visualized by Sypro ruby staining. Bands overrepresented in the pUL34-GST pull-down relative to that with GST were excised and submitted for mass spectrometric analysis at the Biotechnology Resource Center, Cornell University, where the proteins in the gel were digested by trypsin and the masses of derived peptides determined by liquid chromatography-mass spectrometry (LC-MS). Peptides were identified by comparison to the NCBI Human database using MASCOT software (Matrix Science).
In separate experiments, the GST-pUL34 fusion protein bound to glutathione-Sepharose beads was reacted with lysates of uninfected Hep2 cells, and proteins bound to the beads were eluted, electrophoretically separated, and identified by LC-MS as described above.
Immunoblotting.
Nitrocellulose sheets bearing proteins of interest were blocked in 5% nonfat milk plus 0.2% Tween 20 for at least 2 h. The membrane was then probed with lamin A/C mouse monoclonal antibody. Primary antibody was detected by horseradish peroxidase-conjugated bovine anti-mouse secondary antibody (Santa Cruz Biotechnology). All bound immunoglobulins were visualized by enhanced chemiluminescence (Pierce) followed by exposure to X-ray film. Signals were quantified using NIH Image software.
Conventional and immunogold electron microscopy.
Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and 0.25% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium phosphate buffer, pH 7.4, for 30 min at room temperature and then 90 min at 4°C. Cells were washed three times for 5 min each with the same buffer and then dehydrated with a graduated series of ethanol concentrations (10%, 30%, 50%, 70%, and 100%) at 4°C and then −20°C. This was followed by stepwise infiltration with LR-White resin (catalogue no. 14381; Electron Microscopy Sciences) over the course of 48 h at −20°C. Samples were dispensed into gel capsules, and the resin was polymerized at 50°C for 18 h. Thin sections (60 to 90 nm thick) were collected on 300-mesh nickel grids (Ted Pella, Inc., Redding, CA) and floated on drops for the following procedures.
For electron microscopic immunostaining, grids were blocked with 10% normal goat serum and 10% human serum in PBS-0.05% Tween (PBST) and 1% fish gelatin for 15 min at room temperature and were incubated on drops of pUL34-specific chicken antibody diluted 1:100 in PBST plus 1% fish gelatin for 3 h in a humidity chamber at room temperature. After incubation, grids were washed by brief passage over a series of 3 drops in a high-salt buffer (phosphate-buffered 750 mM NaCl, 0.05% Tween, and 1% fish gelatin) and then 5 drops of 1× PBST and fish gelatin. The secondary antibody, donkey anti-chicken immunoglobulin conjugated with 12-nm colloidal gold, was diluted 1:100 in PBST-1% fish gelatin and reacted for 1 h in a humidity chamber at room temperature. The grids were then washed as before on 6 successive drops of PBST-1% fish gelatin and then rinsed in a beaker of 200 ml of filtered water. Grids were air dried at room temperature prior to staining with 2% aqueous uranyl acetate for 20 min and then Reynolds lead citrate for 7 min. Stained grids were viewed in a Philips 201 transmission electron microscope. Conventionally rendered negatives of electron microscopic images were scanned by using a Microtek Scanmaker 5 and Scanwizard Pro PPC 1.02 software. Positive images were rendered from digitized negatives with Adobe Photoshop software.
Conventional electron microscopy was performed as above except that the cells were fixed in 2.5% glutaraldehyde in 0.1 M Na-cacodylate pH 7.4, followed by 2% OsO4 and embedded in Epon-Araldyte resin (EM Sciences).
GST-pUL34 interacts with lamins A and B1 in infected cell lysates.
To identify interaction partners that associated with pUL34, GST or GST fused to the N terminus of full-length pUL34 (GST/pUL34) was affinity purified from Escherichia coli cells. Lysates of uninfected Hep2 cells or Hep2 cells infected with wild-type HSV-1(F) were then reacted with GST or the GST/UL34 fusion protein bound to glutathione-Sepharose beads. Proteins were eluted from the beads in SDS sample buffer, electrophoretically separated on an SDS-polyacrylamide gel, and stained with Sypro ruby. Bands specifically emphasized or unique to the GST/pUL34 reactions as opposed to reactions with GST alone were excised and digested with trypsin, and the masses of peptides were determined by LC-MS. Within a protein band from infected cell lysates corresponding to approximately 70,000 apparent Mr, the masses of 12 tryptic peptides matched tryptic peptides predicted for human lamin A (Table (Table1).1). Moreover, a band containing protein of an apparent Mr of 75,000 from uninfected Hep2 cells contained five peptides consistent with human lamin B1. These data indicate that pUL34 interacts with lamin A/C and lamin B1 in cellular lysates. Although we cannot exclude the possibility that this interaction is indirect, the result combined with our previous observations showing that lamin A and pUL34 interact in rabbit reticulocyte lysates argue for a direct interaction (26).
TABLE 1.
TABLE 1.
Mass spectrometric parameters of lamin tryptic peptides from GST/pUL34 pull-down assays
To determine whether lamin A/C and lamin B1 were specifically pulled down by GST/pUL34, the material pulled down by pUL34/GST and GST alone was electrophoretically separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with either mouse monoclonal antibody directed against lamin A/C or goat polyclonal antibody directed against lamin B (see Materials and Methods). As shown in Fig. Fig.1,1, although some lamin A/C and B1 was bound to Sepharose beads bearing GST alone, the presence of GST-pUL34 increased the amount of bound lamin A and B1 approximately fivefold as revealed by densitometry. In contrast, lamin C was not overrepresented in the GST/pUL34 pull down, despite the fact that lamins A and C were present in roughly equal amounts in the infected cell lysate. These data confirm the specificity of the interaction between pUL34 and lamins A and B1 in cellular lysates and suggest that lamin C is not bound by GST/pUL34.
FIG. 1.
FIG. 1.
Immunoblot of lamin A/C and lamin B pulled down by GST and GST/pUL34. Full-length pUL34 fused to GST or GST alone on glutathione-Sepharose beads was reacted with HSV-1(F)-infected (A) or uninfected Hep2 cell (B) lysates, respectively. Proteins bound to (more ...)
Lamin A/C is required for the proper distribution of pUL34 in infected mouse embryonic fibroblasts.
Given the above data suggesting that lamin A and pUL34 interact in infected cells, we wanted to determine whether the pUL34-lamin A interaction was involved in targeting pUL34 to the nuclear membrane. To test this hypothesis, immortalized LmnA/ MEFs or immortalized MEFs were infected with wild-type HSV-1(F), and the localization of pUL34 was determined by indirect immunofluorescence and confocal microscopy. To assess nuclear shape, the cells were also immunostained with antibody directed against lamin B.
Preliminary experiments indicated that cytopathic effects consequential to HSV infection were accentuated in LmnA/ MEFs as opposed to wild-type MEFs. Specifically, many LmnA/ MEFs underwent dramatic rounding upon infection and subsequently detached from the culture dish by 14 h after infection. By 16 h postinfection, the Lmna/ MEF nuclei were highly distorted in shape, with irregular pUL34 staining in punctate foci that associated with the nuclear rim (data not shown). Because the exaggerated changes in nuclear shape at late times after infection might affect pUL34-specific localization indirectly, the cells were fixed at 13 hours postinfection when effects on nuclear shape were less prominent. The cells were then permeabilized and immunostained with antibodies directed against pUL34 and lamin B. The results are shown in Fig. Fig.22.
FIG. 2.
FIG. 2.
Indirect immunofluorescence localization of pUL34 in HSV-1 infected MEFs containing or lacking lamin A/C. Control MEFs or Lmna/ MEFs were infected with either wild-type HSV-1(F) (A to F) or Us3 kinase-dead mutant virus (K220A) (G to (more ...)
Optical slices recorded from points midway between the top and adherent surface of the cells revealed that pUL34-specific immunostaining localized at the nuclear rim of LmnA/ cells, despite the absence of lamin A. On the other hand, the smooth distribution of pUL34 seen in infected MEFs was not observed in cells lacking lamin A/C. Rather, considerable areas of the nuclear rim of LmnA knockout cells were stained with lamin B-specific antibody but lacked detectable pUL34-specific immunostaining. Other regions of the nuclear rim contained areas with intense pUL34 immunostaining, and these regions often overlapped lamin B immunostaining. We conclude that while lamin A/C is dispensable for localization of pUL34 at the nuclear rim, its presence is required for the normal smooth localization of pUL34 around the nuclear rim.
Given that lamin A/C was required for the normal distribution of pUL34 throughout the nuclear rim, we next wanted to determine whether lamin A/C was necessary for localization of pUL34 at the INM. To test this possibility, LmnA/ or wild-type MEFs were infected with HSV-1(F), and thin sections of the infected cells were reacted with pUL34-specific IgY followed by reaction with 12-nm colloidal gold-conjugated anti-IgY. As shown in Fig. Fig.3,3, pUL34-specific immunoreactivity was associated with both the inner and outer nuclear membranes of both cell types (Fig. 3A to C). We therefore conclude that lamin A/C is not required for targeting pUL34 to the INM.
FIG. 3.
FIG. 3.
Ultrastructural localization of pUL34 in MEFs or LmnA/ MEFs infected with HSV-1(F) or US3 kinase-dead virus. (A) Wild-type MEFs were infected with HSV-1(F), and localization of pUL34 was determined by reaction with pUL34-specific IgY (more ...)
To determine the effects of lamin A/C on HSV-1 replication, LmnA/ and wild-type MEFs were infected with 5.0 PFU/cell of wild-type HSV-1(F), and the virus infectivity was measured at various times after infection. The results, shown in Fig. Fig.4,4, indicated very little difference in the amount of infectious virus produced from the two cell types at all time points. Thus, lamin A/C neither enhanced nor hindered production of wild-type HSV-1(F), at least at high multiplicities of infection.
FIG. 4.
FIG. 4.
One-step growth curve of HSV-1 on normal MEFs or LmnA/ MEFs. Cells were infected with either HSV-1(F) or Us3(K220A) at 5 PFU per cell. Residual infectivity was inactivated by a low-pH wash at 1 hour postinfection. At the indicated time (more ...)
Effects of US3 kinase activity on viral infectivity and localization of pUL34 in the presence and absence of lamin A/C.
The US3 kinase phosphorylates lamin A/C and pUL34, promotes production of infectious virus, and optimizes nuclear egress of virions. To determine whether the effects of US3 kinase activity on nuclear egress and pUL34 localization were dependent on lamin A/C, LmnA/ or wild-type MEFs were infected either with a virus containing a mutation in US3 (K220A) that precludes kinase activity or with wild-type HSV-1(F) virus. As shown in Fig. 2G to L, and consistent with results from other cell lines, both cell types exhibited a distribution of pUL34 that differed from that of cells infected with the virus expressing active US3 kinase. Specifically, pUL34 localized in punctate regions at the nuclear rim, as opposed to the more diffuse distribution seen in the presence of US3 kinase activity. On the other hand, the position of the pUL34-containing foci in LmnA/ cells was most often on the cytoplasmic side of the nuclear rim, whereas these foci were located mostly on the nucleoplasmic side of the nuclear rim in normal MEFs. The latter distribution resembled pUL34 localization in other cell lines examined previously (30). Thus, although lamin A/C is not required for the punctate distribution of pUL34 at the nuclear rim of cells infected with the US3 kinase-dead virus, it is required to retain these pUL34-containing foci within the nucleoplasm.
To more precisely analyze the effects of US3 kinase activity on virion egress in the presence or absence of lamin A/C, thin sections of LmnA knockout MEFs and normal MEFs infected with wild-type and US3 kinase-dead viruses were examined by electron microscopy. As shown in Fig. Fig.3D,3D, virions accumulated aberrantly in pockets of nuclear membranes of cells infected with the US3 kinase-dead virus in normal MEFs. In most cases, these virion-containing regions were located internal to the nuclear rim. The appearance of infected lamin A knockout cells differed substantially in three respects. First, membranous structures that appeared to represent extensions of the INM were often observed (Fig. (Fig.3G3G and inset). Secondly, the space between the INM and ONM was often exaggerated relative to uninfected cells and cells infected with wild-type HSV-1(F). Third, virions were not observed to accumulate to a large extent in the perinuclear space, even upon infection with the US3 kinase-dead virus. We conclude that lamin A is required for (i) the accumulation of virions in discrete regions of the perinuclear space normally seen in cells infected with the US3 kinase-dead virus and (ii) the morphology of the INM normally seen in infected cells.
To determine whether the concentrations of pUL34 as revealed by immunofluorescence reflected localization of pUL34 within the extensions of the INM observed in LmnA knockout cells, we performed immunogold electron microscopy on these cells infected with the US3 kinase-dead virus. Whereas immunoreactivity with pUL34-specific antibody was not detected in any cell line infected with the UL31 null virus (data not shown), extensions of INM were heavily decorated with gold beads reflecting the localization of pUL34 (Fig. (Fig.3G,3G, inset). Thus, pUL34 localization in nuclear membrane extensions correlated with the punctate distribution of lamin A knockout cells infected with the US3 kinase-dead virus.
To determine the effects of lamin A/C and US3 kinase activity on viral infectivity, LmnA/ MEFs or wild-type MEFs were infected with US3(K220A) or HSV-1(F) virus at 5.0 PFU/cell. At various times after infection the cells were lysed and the amount of infectious virus was determined by plaque assay on Vero cell monolayers. As shown in Fig. Fig.4,4, the amount of virus produced by US3(K220A) was reduced by at least 10-fold at all times relative to the amount produced by HSV-1(F) regardless of whether the cells produced lamin A/C or not. Perhaps the most striking effect, as noted in previous studies (28, 30), was a delay in the onset of production of infectious virus in the absence of US3 kinase activity, resulting in a reduction in viral titer by more than 1,000-fold at 6 h postinfection. This delay occurred whether or not lamin A/C was produced. These data indicate that US3 kinase activity enhances both early and late production of infectious virus independently of its effects on lamin A/C.
Pertaining more to the purposes of this report was the observation that infection of LmnA/ cells with the US3(K220A) virus yielded approximately 10-fold more infectious virus at 12 h postinfection relative to the amount produced in wild-type MEFs. The discrepancy in titer decreased later in infection, such that at 18 and 24 h after infection infectious virus produced by the LmnA knockout cells was increased only fivefold and threefold, respectively, over that obtained from normal MEFs. These data indicate that expression of lamin A/C decreases production of infectious virus and that US3 kinase activity partially relieves the lamin A/C-dependent impediment to viral replication.
Lamin B1 is dispensable for localization of pUL34 to the nuclear rim but is required for normal viral growth.
As shown in Table Table11 and noted above, peptides in the pUL34/GST pull-down assay were consistent with the predicted masses of tryptic peptides derived from four different regions of lamin B1. To determine whether the interaction between pUL34 and lamin B was specific, the proteins pulled down with pUL34/GST or GST were subjected to immunoblotting with lamin B-specific antibody. As shown in Fig. Fig.1B,1B, lamin B-specific immunoreactivity was readily detected in the pUL34/GST pull-down assay, confirming the mass spectrometric analysis. Moreover, densitometry revealed that fivefold less immunoreactivity was detected in the material pulled down with GST. Thus, we conclude that the interaction with lamin B1 was specific, although we could not exclude the possibility that the interaction was indirect.
Given the potential interaction between pUL34 and lamin B1, we determined whether lamin B1 plays a role in localization of pUL31 and pUL34 to the nuclear membrane by using a similar strategy as employed in the study of lamin A. Thus, MEFs lacking lamin B1 were obtained and infected with 5.0 PFU HSV-1(F) or US3(K220A) per cell, and the localization of pUL34 was determined by indirect immunofluorescence 13 h later. As shown in Fig. Fig.5,5, and unlike the effects caused by the absence of lamin A/C, the distribution of pUL34-specific immunostaining in the lamin B knockout cells was not visibly altered from that seen in wild-type MEFs. On the other hand, HSV-induced cytopathic effects were accentuated in the lamin B1 knockout cells compared with infected normal MEFs. The alteration in cell morphology as a consequence of virus infection was severe enough to pose difficulties in maintaining the lamin B knockout cells on coverslips late after infection (data not shown).
FIG. 5.
FIG. 5.
Confocal indirect immunofluorescence determination of the distribution of pUL34 in HSV-1 infected MEFs containing or lacking lamin B1. Wild-type MEFs (A and C) or LmnB1/ MEFs (B and D) were infected with HSV-1(F) or Us3(K220A) virus, (more ...)
To determine the effects of the US3 kinase on distribution of pUL34 in the absence of lamin B1, the LmnB1 knockout cells and wild-type MEFs were infected with the US3(K220A) virus and the localization of pUL34 was determined by indirect immunofluorescence. As shown in Fig. Fig.5,5, pUL34 localized in a similar punctate distribution at the nuclear rim of both cell lines. On the other hand, the foci in the lamin B1 knockout cells appeared less numerous than those observed in the wild-type MEFs infected with the US3 kinase-dead virus.
As shown in Fig. 6C, D, and E, representative electron microscopic images of many cells examined revealed that, unlike the case in the lamin A knockout cells, lamin B1 knockout cells infected with the US3 kinase-dead virus contained regions of nuclear membrane bearing virions. A feature unique to these cells infected with this virus was the presence of very densely staining scalloped-shaped extensions of what appeared to be the nuclear membrane (Fig. 6D and E). These observations taken together with the indirect immunofluorescence studies indicate that lamin B1 is dispensable for targeting of pUL34 to the nuclear rim of infected MEFs, but it is required for normal nuclear membrane morphology of cells infected with the US3 kinase-dead virus.
FIG. 6.
FIG. 6.
Localization of pUL34 in LmnB/ cells infected with HSV-1(F) or Us3(K220A) viruses. (A) Low magnification of an HSV-1(F)-infected cell. (B) Section of nuclear membrane of cell infected with HSV-1(F). Positions of gold beads indicative (more ...)
Immunogold electron microscopy experiments were also performed to determine the localization of pUL34 in the lamin B1 knockout cells. As shown in Fig. Fig.6B,6B, gold beads indicative of the localization of pUL34 localized at the nuclear rim of cells infected with the wild-type virus. Moreover, pUL34-specific immunoreactivity was noted at the INM of lamin B knockout cells infected with the US3 kinase-dead mutant. This immunostaining localized mostly within the densely staining, scalloped-shaped regions of the nuclear membrane or was associated with nearby virions (Fig. 6D and E). These data therefore indicate that lamin B1 is dispensable for targeting pUL34 to the INM but necessary for the normal morphology of the nuclear membrane in the absence of US3 kinase activity.
We next examined the effects of the absence of lamin B1 on viral infectivity in the presence and absence of the US3 kinase. As shown in Fig. Fig.7,7, the lamin B1 knockout cells were less permissive to HSV replication than normal MEFs, regardless of whether the US3 kinase was active. Specifically, titers at all time points in wild-type MEFs were at least 10-fold greater than those reached in the lamin B1 knockout cells infected with either wild-type virus or the US3 mutant virus. We therefore conclude that lamin B1 contributes substantially to the production of infectious virus, but this does not reflect a role in targeting pUL34 to the INM.
FIG. 7.
FIG. 7.
Growth curves of HSV-1(F) or US3 kinase-dead virus in MEFs or LmnB/ MEFs. The experiment was performed similarly to that described in the legend to Fig. Fig.44.
Taken together, the data presented herein indicate that targeting of pUL34 to the INM and aggregation in the absence of Us3 kinase activity are not dependent on expression of lamin A/C or B1, despite the observation that these proteins were shown to interact with pUL34 in infected cell lysates. On the other hand, the absence of lamin A/C altered the normal smooth distribution of pUL34 as revealed by indirect immunofluorescence, suggesting that it is directly or indirectly involved in pUL34 distribution within the nuclear rim. In contrast, lamin B1 was entirely dispensable for pUL34 targeting to the nuclear rim, as viewed by indirect immunofluorescence, and to the INM, as viewed by immunogold electron microscopy. Thus, lamin A or lamin A-interacting components of the nuclear lamina are more relevant to pUL34 targeting than lamin B1 or lamin B1-interacting components. The effects of lamin A on pUL34 targeting may reflect effects of lamin A on lamina mechanics and stiffness, functions which are largely independent of lamin B1 (16). Alternatively, data obtained in this study and in previous work in which lamin A, pUL31, and pUL34 were shown to interact in pull-down assays from rabbit reticulocyte lysates suggest that the lamin A-pUL34 interaction is direct and may play a role in pUL34 targeting (26). The data are also consistent with the observation that emerin, a lamin A-interacting protein, can interact with pUL34 and is displaced in HSV-infected cells in a UL34-dependent manner (17). Whether the emerin-pUL34 interaction is mediated through interactions with lamin A/C will require further studies. A final possibility is that lamin B1 is involved in INM targeting of pUL34, but lamin A confers redundant functions that compensate for the absence of lamin B1.
An untested but widely accepted hypothesis is that the nuclear lamina poses a barrier that herpesviruses must breach to allow nucleocapsids access to the INM for envelopment. Along these lines, previous observations indicated that the lamina is disrupted in HSV-infected cells in a UL31/UL34-dependent manner (2, 23, 26, 33, 34). Several studies have identified mechanisms by which pUL31/pUL34 of HSV may accomplish nuclear lamina disruption. Specifically, protein kinase Cδ and -α have been shown to be recruited to the nuclear membrane in a pUL31/pUL34-dependent manner to augment lamin B phosphorylation, whereas US3 has been shown to phosphorylate lamin A directly in vitro and to be required for its optimal phosphorylation in infected cells (23, 24). Moreover, pUL31/pUL34 may have their own lamina-depolymerizing activities inasmuch as overexpression of these proteins in the absence of other proteins are sufficient to disrupt the lamina, and locally high concentrations of pUL31/pUL34 exaggerate adjacent lamina perforations (2, 23, 26, 33). The observation in this study that lamin A knockout cells are more permissive to replication of a US3 kinase-dead virus at high multiplicities of infection further argues that at least lamin A poses a barrier to replication (and presumably nucleocapsid envelopment) and that US3 helps to overcome this barrier. Thus, disruption of the lamina might indeed reflect an important role for these proteins in promoting virion budding at the INM.
Despite their relative permissiveness to virus replication at high multiplicities of infection, the lamin A knockout cells exhibited altered NM morphology when infected with the US3 kinase-dead virus. Specifically, exaggerated extensions of the INM and increased space between the INM and ONM were noted. Because these effects were precluded by US3 kinase activity, it follows that substrates of US3 kinase other than lamin A are responsible for these phenotypes. Previous studies reported increased spacing between the INM and ONM upon overexpression of UL34, pUL31/pUL34-dependent alterations of the nuclear lamina, and budding from the INM upon coexpression of pUL31 and pUL34 of pseudorabies virus in the absence of capsids (14, 41). Thus, the effects on nuclear membrane morphology induced by the combined absence of US3 kinase activity and lamin A might be consequential to increased concentration of pUL34/pUL31 in certain regions of the INM. This is consistent with the observation by immunogold electron microscopy that pUL34 was specifically detected in the unusual nuclear membrane extensions observed in this study.
Also interesting to us was the absence of virions trapped within the perinuclear space of the LmnA knockout MEFs infected with the US3 kinase-dead virus, despite the fact that this feature was commonplace in similarly infected normal MEFs. Thus, lamin A is somehow required for the observed retention of virions in discrete regions within the perinuclear space. We speculate that this reflects the general permissivity of primary envelopment in the lamin A knockout cells. Thus, capsids may bud through the INM at multiple sites in the absence of lamin A, whereas the position of envelopment sites are more restricted by the lamin A-imposed barrier in normal MEFs. The absence of US3 kinase activity would still delay fusion of the virion envelope and ONM, but the wide dispersal of virions in the perinuclear space of lamin A knockout cells would preclude virion aggregation. Alternatively, we cannot rule out the possibility that the nuclear envelope is generally more fragile in the lamin A knockout cells, thus precluding stable association of virions within the perinuclear space of cells as they are pelleted prior to fixation for electron microscopy.
We also noted that pUL34-containing regions mostly localized external to the nuclear membrane in lamin A knockout cells infected with the US3 kinase mutant but internal to the nuclear membrane in MEFs or lamin B knockout cells infected with this virus. We speculate that lamin A, by enhancing lamina stiffness, restrains locally high concentrations of the pUL31/pUL34 complex from inducing cytoplasmic protrusions of the nuclear membrane, although we cannot exclude more indirect mechanisms.
In striking contrast to the results with lamin A, LmnB1 knockout MEFs were less permissive to viral replication at high multiplicities of infection, and this was observed in both the presence and absence of US3 kinase activity. The role of lamin B1 in HSV infection is unclear, but it is required for a number of important nuclear functions including transcription, DNA replication, cell signaling, and optimal reassembly of the nucleus after mitosis (36). Thus, disturbance of any of these or related functions might render the cells less permissive by impairing the machinery that the virus ultimately needs to commandeer for optimal infection. This may be one reason why alteration of the nuclear lamina during viral infection is mostly limited to very discrete regions. Such a strategy might limit detrimental effects on the cell while still allowing nucleocapsids to bud through the INM.
Acknowledgments
We thank Richard Roller for the US3 kinase-dead virus and pUL34 antibody, Karen Reue for the lamin B knockout and control MEFs, and Colin Stewart for the lamin A knockout MEFs.
These studies were supported by RO1 grant AI52341 from the National Institutes of Health.
Footnotes
[down-pointing small open triangle]Published ahead of print on 4 June 2008.
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