ARTICLE |
Correspondence to: Helle L. Jensen, the Protein Laboratory, the DNA Tumour Virus Group, University of Copenhagen, The Panum Institute, Bldg. 6.2., Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Herpes simplex virus type 1 (HSV-1) is a common human pathogen of skin and mucous membranes and is potentially dangerous when the infection is disseminated. Viral morphogenesis, especially the mechanism of viral envelopment and the exact pathway for processing and transport of HSV-1 glycoproteins, is still unclear. We report the results of optimized immunogold-labeled cryosection electron microscopy of HSV-1-infected cultured human fibroblasts (MRC-5). The simplified method presented has proved necessary to obtain reproducible results on cellular distribution of viral glycoproteins. It is now possible to demonstrate the viral glycoprotein gD-1, but not gC-1, in the nuclear membranes and to demonstrate gD-1- and gC-1-labeled viral particles in the perinuclear space, and to show the fate of the viral particles in the endoplasmic reticulum and Golgi area in infected cells. (J Histochem Cytochem 46:487496, 1998)
Key Words: cryosection, electron microscopy, glycoproteins, HSV-1, immunogold
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The viruses are small biological structures that can be observed only in the electron microscope. Not having their own metabolism, viruses are obligate parasites, which is why viruscell interactions are of both viral and cell biological interest. Herpes simplex virus Type 1 (HSV-1) is an enveloped virus with a double-stranded DNA genome, which encodes at least 15 membrane proteins including 11 identified glycoproteins (
With the light microscopic method, one can state only that the HSV-1 glycoproteins are located near the nucleus, in the cytoplasm, or in the plasma membrane (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents, Antibodies, and Conjugates
All chemicals were acquired from Merck (Darmstadt, Germany) unless otherwise indicated.
The monoclonal antibodies (MAbs) against HSV-1 gD (MW 60,000) glycoprotein Fd 138-80 (-light chains (no. N 357), goat anti-mouse (GAM-G5) IgG (no. RPN 424) and goat anti-rabbit (GAR-G5) IgG (H+L) conjugated to 5-nm colloidal gold particles (no. RPN 420) plus goat anti-rabbit (GAR-G1) IgG (H+L) conjugated to 1-nm colloidal gold particles (no. RPN 470) were purchased from Amersham (Poole, UK). The actual probe diameter of AuroProbe One is 78 nm and of AuroProbe EM G5 is 1215 nm (
(no. M 742), affinity-purified (
General Remarks
All experiments were repeated at least twice and duplicates of both grids and slides were made. In each experiment, 200300 cells were examined in the electron microscope and the results were scored semiquantitatively.
The optimal working dilution and incubation time in a humid chamber at room temperature (RT) were determined for all batches of antisera and conjugates. Gold conjugates were titrated by anti-ß-tubulin immunostaining of non-virus-infected cells (MOCK). Antisera were stored at -80C, avoiding repeated thawing and freezing. Dilutions of antisera and conjugates were made just before use. PBS (10 mM Na2HPO4/KH2PO4, 150 mM NaCl, pH 7.4) and PBS-buffered solutions were renewed every second week. Glycine and bovine serum albumine (BSA) were stored at -20C for 6 months. Sucrose, methyl cellulose, and aqueous uranyl acetate were renewed every fourth week and stored at 4C. Great care was taken in preparing solutions, and attention was directed to controlled temperature conditions through all procedures.
Quality control of the immunogold-labeled ultrathin sections for electron microscopy included preservation of ultrastructural morphology, no precipitation or clumping of the gold probe, and minimal but just visible background staining with few gold particles over the nuclei to ensure saturation of the antigenantibody reaction. Further, it was required that cross-sectioned tubulin in MOCK cells was labeled with about 10 G5 particles and that longitudinal sectioned tubulin showed gold particles with a linear disposition.
Light Microscopy
Conditions for the low-fading immunofluorescence indirect labeling with propidium iodide contrast in whole cells and semithin cryosections have been described earlier (
Silver Enhancement
Silver amplification with IntenSE M (no. RPN 491; Amersham) was performed as set out in the pack leaflet at 18C in a darkroom under red safelight and was compared to physical development (
Serum
The goat serum (State Serum Institute; Copenhagen, Denmark) and FBS (fetal bovine serum sterile, no. 29-101-54; Flow Laboratories, McLean, VA) were decomplemented by heat inactivation for 30 min at 56C (
Viral Stock and Titration Assay
Viral stocks of HSV-1 strain F (
The viral titers of the viral stocks were determined by plaque assay (
Cell Preparations
MRC-5 delivered at passage 2628 were grown and proved mycoplasma-free as described previously (
To make frozen cell cultures, the cells were scraped off the culture flasks with a plastic policeman, then embedded in gelatin without air bubbles, and after 30 min at 37C centrifuged at 12,000 rpm for 5 min. After 1 hr at 4C, the pellet was cut with razor blades into cubes about 1 mm3 and cryoprotected in 2.3 M PBS-buffered sucrose (no.7651; Merck) with 1% PFA overnight for roughly 18 hr at 4C, frozen, and stored in liquid nitrogen (
Semithin (about 500 nm thick) cryosections for light microscopy and ultrathin (about 100 nm thick) cryosections for electron microscopy were cut with a glass knife in an RMC MT 6000 XL cryoultramicrotome. The sections were removed from the knife with aid of a sucrose droplet in a wire loop (
Optimized Immunogold Labeling
The ultrathin cryosections on the grids were transferred by aid of the smallest possible 0.25-mm platinum wire loop through great drops on a sheet of Parafilm, as follows. In addition to washing (twice for 5 min) with PBS, pH 7.4, glycine (no. 4201; Merck) 0.02 M in PBS was put on for 10 min to inactivate any remaining aldehyde. After washing with PBS (twice for 5 min), PBS containing 0.8% BSA (A 4503; Sigma Chemical) and 0.1% IGSS quality gelatin followed for 5 min. Thereafter, incubation was carried out for 45 min with the primary antibody (Fd 138-80 1:250, DL6 1:1500, 1D3 1:125, HD1 1:1000, HC1 1:800 or anti-ß-tubulin 1:6001:1000 in different batches) diluted in PBS + BSA + gelatin solution with 1% goat serum. The primary antibody was washed off with PBS + BSA + gelatin solution (six times for 4 min). In the indirect three-layer (GAR-G5 or GAR-G1) but not in the two-layer (GAM-G5) labeling method, this was followed by incubation for 30 min with affinity-purified RAM 2.5 µg/ml in PBS + BSA + gelatin solution containing 1% goat serum and subsequent washing with PBS + BSA+ gelatin solution (six times for 4 min). Incubation followed for 20 min with GAR-G1, GAR-G5 or GAM-G5 in suitable concentrations of 1:251:75 diluted in PBS + BSA + gelatin buffer with 1% goat serum. Washing followed with PBS + BSA+ gelatin buffer (six times for 4 min), PBS, pH 7.4 (six times for 4 min), and postfixation with 2% GA in PBS, pH 7.4 (10 min). An intervening wash was performed with PBS, pH 7.4 (four times for 4 min), and glass-distilled water (four times for 4 min). If desired, the silver enhancement of the gold particles was done for 8.5 min, followed by washing in glass-distilled water three times for 5 min. The sections were then stained and protected against air-drying artifacts (
Controls
Immunocytochemical controls were carried out by (a) immunostaining of MOCK cells, (b) omission of the primary or secondary antibody and replacement with buffer or type-matched unrelated MAb UCHL1 1:25, or (c) with anti-ß-tubulin serving as a positive control. The specificity of the MAbs and the reliability of the immunogold labeling were controlled by immunoblotting and immunofluorescence light microscopy.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increasing the MOI from 10 (50% infected cells) to 30 (90% infected cells) caused diffuse labeling rather than infection visible in spots of the monolayer cell culture. Electron microscopic studies investigate only a minor part of the sample, which is why the most convenient MOI of 30 was chosen for future studies. Furthermore, the labeling intensity appeared to increase with rising MOI. Exceeding MOI 30 was risky because the cells tended to fall off the culture flask before the time for harvesting.
Morphology
The MOCK cells were long and slender, with regular nuclei and insignificant vacuolization of the cytoplasm (Figure 1F). Infection with HSV-1 rendered the cell larger, with variable rounding (Figure 1AE), the nuclei became irregular with margination of the nuclear chromatin, and the cytoplasm was vacuolated in part, caused by dilatation of the endoplasmic reticulum and the Golgi sacs (Figure 4). The membranous ultrastructure was well preserved (Figure 2 and Figure 3).
|
|
Fixation
Compared to 8% PFA, the impact of adding GA to the fixative consisted of improved ultrastructure and, to some extent, reduced intensity of the specific labeling, especially at the plasma membrane. Basically, the labeling was independent of the applied concentrations of GA. Therefore, for further investigations, the fixative of 3% PFA added 2% GA was preferred.
Embedment
The 20% Merck gelatin was preferable for embedment because it yielded vivid labeling, because the ultrastructure was uniform in the compared gelatins, and because it was difficult to cut samples embedded in the other gelatins. In fact, chatter was now the only troublesome technical problem.
Immunolabeling
The simplified cell preparation and immunoincubation instructions using only PBS-buffered solutions described were reproducible, and cryopreparations have been stored in liquid nitrogen for 5 years without losing immunoreactivity. Specific labeling of HSV-1 glycoproteins was observed, although to a lesser degree, by the two-layer indirect immunogold method (Figure 3) compared to the three-layer technique if the concentration of the primary antibody was doubled and the incubation time with GAM increased to 60 min. No further significant labeling was obtained by higher concentrations, longer incubation times at room temperature, incubation overnight at 4C, or incubation according to the scheme but at 37C. Compared to the 5-nm gold, the 1-nm gold particles (Figure 4) intensified the labeling but did not provide new information. The enhanced particles vary in size and shape, and there is a risk for producing artifacts.
Glycoproteins
The 1D3 antibody worked in a three-layer indirect immunofluorescence labeling of 8% PFA-fixed cells (Figure 1E), but convincing ultrastructural specific labeling was never observed. Thus far, we have not seen different localization of the labeling of the discontinuous epitope visualized by HD1 and the continuous gD-1 epitope labeled by the antibody DL6. Optimized immunolabeling of cryosections allowed the identification of both gC-1 (Figure 6) and gD-1 on the surface of extracellular HSV-1 viral particles, on the plasma membrane (Figure 1A, Figure 1CE, Figure 6, and Figure 8), on the surface of intracellular enveloped viral particles (Figure 4), in the Golgi apparatus (Figure 4), and in the endoplasmic reticulum (Figure 7). It was possible to demonstrate both gD-1 and gC-1 in the cell adhesion areas and sometimes on the surface of enveloped viral particles localized in the perinuclear space (Figure 5 and Figure 7). However, distinct labeling of the nuclear membranes was demonstrated only with the gD antibodies (Figure 1B and Figure 5). Capsids were seen close to and in the nuclear membranes (Figure 3), but complete budding and evidential acquirement of envelope from the nuclear membranes were never detected. The fate of enveloped and unenveloped viral particles in the endoplasmic reticulum (Figure 8) and in the Golgi area (Figure 4) could be followed.
|
Controls
Immunostaining of MOCK cells (Figure 1F) and omission of the primary or secondary antibody in HSV-1-infected cells were all gD-1- and gC-1-negative.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are three essential problems in immunoelectron microscopy of Herpes simplex-virus-infected cells: (a) the number of labeled cells, because only minor areas are investigated; (b) the intensity of immunostaining; and (c) reproducibility of immunolabeling. An ultrathin section of 100 nm represents approximately 1% of the whole-cell antigenicity (calculated from Figure 2), which calls for special considerations. Moreover, the results can be influenced by the thickness of the sections, the infection of different cells is not synchronized, and even the most optimized procedure allows antigens to be destroyed to various degrees. Therefore, we do not find quantitative analysis of the labeling possible.
Previous immunogold labeling studies of HSV-1-infected human embryonic lung cells processed for ultrathin cryosections (
Ultrastructural studies, in particular, need fixation to preserve the cell structure and to immobilize soluble antigens. Unfortunately, fixatives tend to modify or abolish antigenicity by alteration of the primary, secondary, or tertiary structure of antigens. In accordance with
The advantages of colloidal gold markers at the electron microscopic level include the electron density, the stability, and the facts that it is easy to prepare in different and even small diameters and is easy to recognize (
One of the limitations of electron microscopy is sampling, wherein only a small proportion of the cells can be studied. The electron microscopic examinations revealed areas of cells with extraordinarily high levels of capsids near the nuclear membrane, viral particles in the perinuclear space, gD-1 labeling of the nuclear membranes, or well-preserved endoplasmic reticulum. In addition, there is cell-to-cell variation of the infection and in different levels of the sections, which makes it necessary to study many cells. By examining 200300 cells at passages below 35 in each experiment, we obtained uniform results from the method used.
To the best of our knowledge, our observation by electron microscopy is the first demonstration of gD-1 in the nuclear membranes and on enveloped viral particles located in the perinuclear space of HSV-1-infected human fibroblasts by immunogold labeling of cryosections. Several conventional and immunoelectron microscopic studies on the morphogenesis of Herpes simplex-infected cells have been carried out over the past three decades. Surface epitopes have been examined by immunolabeling and negative staining of unembedded viral particles (
In theory, the ability to identify discontinuous epitopes in ultrathin cryosections should be reduced compared to continuous epitopes. Thus far, we have observed only a quantitative but not a qualitative disparity between labeling with the antibody HD1 to a discontinuous gD-1 epitope and the antibody DL6 to a continuous gD-1 epitope. The presence of HC1 antigen in the nuclear membranes might be at a density below the level of detection with the reagents and the technique used, either because of destruction or because of minimal production or excessive release. It has previously been reported that envelopment occurs at the thickened or reduplicated nuclear membrane (
![]() |
Acknowledgments |
---|
Supported by grants from the Danish Cancer Society, The Foundation of Health Insurance "Danmark," Erik Hørslev and wife Birgit Hørslev's Fund, the Else and Mogens WedellWedellsborgs Fund, the DMA Research Fund, the Novo Nordisk Foundation, the Danish Foundation for Advancement of Medical Science, and the Aage Bang's Foundation.
We gratefully acknowledge the support of Prof O. Norén, Prof H. Sjöström, and Assoc Prof G.H. Hansen (University of Copenhagen, Denmark) in putting cryosection and electron microscopy facilities at our disposal. Generous donations of monoclonal antibodies DL6 and 1D3 were given by Dr R.J. Eisenberg and Dr G.H. Cohen (University of Pennsylvania, Philadelphia, PA), Fd 138-80 by Dr S. Chatterjee (University of Alabama, Birmingham, AL), and HC1 and HD1 by Dr L. Pereira (University of California, San Francisco, CA). We would like to thank J. GrøndahlHansen, PhD, for affinity-purification of rabbit anti-mouse immunoglobulin. L.M. Leerhøj, N. Broholm, and L. Bæhr rendered excellent technical assistance.
Received for publication June 9, 1997; accepted November 4, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avitabile E, Ward PL, Lazzaro CD, Torrisi MR, Roizman B, CampadelliFiume G (1994) The Herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus. J Virol 68:7397-7405[Abstract]
Baines JD, Jacob RJ, Simmerman L, Roizman B (1995) The Herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells. J Virol 69:825-833[Abstract]
Danscher G (1981) Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electronmicroscopy. Histochemistry 71:1-16[Medline]
Eisenberg RJ, Long D, Pereira L, Hampar B, Zweig M, Cohen GH (1982) Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of Herpes simplex virus type 1. J Virol 41:478-488[Medline]
Eisenberg RJ, Long D, Ponce de Leon M, Matthews JT, Spear PG, Gibson MG, Lasky LA, Berman P, Golub E, Cohen GH (1985) Localization of epitopes of Herpes simplex virus type 1 glycoprotein D. J Virol 53:634-644[Medline]
Ejercito PM, Kieff ED, Roizman B (1968) Characterization of Herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol 2:357-364[Medline]
Friedman HM, Cohen GH, Eisenberg RJ, Seidel CA, Cines DB (1984) Glycoprotein C of Herpes simplex virus 1 acts as a receptor for the C3b complement component on infected cells. Nature 309:633-635[Medline]
Gilbert R, Ghosh K, Rasile L, Ghosh HP (1994) Membrane anchoring domain of Herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization. J Virol 68:2272-2285[Abstract]
GrøndahlHansen J, Ralfkiær E, Nielsen LS, Kristensen P, Frentz G, Danø K (1987) Immunohistochemical localization of urokinase- and tissue-type plasminogen activators in psoriatic skin. J Invest Dermatol 88:28-32[Abstract]
Hopwood D (1985) Cell and tissue fixation, 1972-1982. Histochem J 17:389-442[Medline]
Isola VJ, Eisenberg RJ, Siebert GR, Heilman CJ, Wilcox WC, Cohen GH (1989) Fine mapping of antigenic site II of Herpes simplex virus glycoprotein D. J Virol 63:2325-2334[Medline]
Jensen H, Broholm N, Norrild B (1995a) Low-fading immunofluorescence with propidium iodide contrast compared with immunogold light microscopy in whole cells and semi-thin cryosections. J Histochem Cytochem 43:507-513
Jensen HL, Rygaard J, Norrild B (1995b) A time-related study of brefeldin A effects in HSV-1 infected cultured human fibroblasts. APMIS 103:530-539[Medline]
Koga J, Chatterjee S, Whitley RJ (1986) Studies on Herpes simplex virus type 1 glycoproteins using monoclonal antibodies. Virology 151:385-389[Medline]
Komuro M, Tajima M, Kato K (1989) Transformation of Golgi membrane into the envelope of Herpes simplex virus in rat anterior pituitary cells. Eur J Cell Biol 50:398-406[Medline]
Larsson L-I (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL, CRC Press
Larsson L-I (1993) Tissue preparation methods for light microscopic immunohistochemistry. Appl Immunohistochem 1:2-16
La Thangue NB, Chan WL, Almeida JD (1984) Monoclonal antibodies to Herpes simplex virus type 1 glycoproteins show that epitope location influences virus neutralization. J Med Virol 13:227-242[Medline]
Lausch RN, Staats H, Oakes JE, Cohen GH, Eisenberg RJ (1991) Prevention of herpes keratitis by monoclonal antibodies specific for discontinuous and continuous epitopes on glycoprotein D. Invest Ophthalmol Vis Sci 32:2735-2740[Abstract]
Leunissen JLM, van de Plas PFEM, Borghgraef PEJ (1989) AuroProbe One: a new and universal ultra small gold particle based (immuno)detection system for high sensitivity and improved penetration. AuroFile (Janssen, Olen, Belgium) 2:1-2
LopezIglesias C, PuvionDutilleul F (1988a) Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection. Biol Cell 62:47-56[Medline]
LopezIglesias C, PuvionDutilleul F (1988b) Visualization of glycoproteins after tunicamycin and monensin treatment of Herpes simplex virus infected cells. J Ultrastruct Mol Struct Res 101:75-91[Medline]
Nielsen MH, Bastholm L, Chatterjee S, Koga J, Norrild B (1989) Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections. Histochemistry 92:89-93[Medline]
Nii S, Morgan C, Rose HM (1968a) Electron microscopy of Herpes simplex virus. II. Sequence of development. J Virol 2:517-536[Medline]
Nii S, Morgan C, Rose HM, Hsu KC (1968b) Electron microscopy of Herpes simplex virus. IV. Studies with ferritin-conjugated antibodies. J Virol 2:1172-1184[Medline]
Norrild B, Lehto V-P, Virtanen I (1986) Organization of cytoskeleton elements during Herpes simplex virus type 1 infection of human fibroblasts: an immunofluorescence study. J Gen Virol 67:97-105[Abstract]
Norrild B, Nielsen MH, Bastholm L, Chatterjee S (1991) Intracellular maturation and sorting of two Herpes simplex virus type 1 glycoproteins. Immunogold staining of ultrathin cryosections. APMIS 99:371-380[Medline]
Pereira L, Klassen T, Baringer JR (1980) Type-common and type-specific monoclonal antibody to Herpes simplex virus type 1. Infect Immun 29:724-732[Medline]
Pietschmann SM, Gelderblom HR, Pauli G (1989) Compartment-specific immunolocalization of conserved epitopes of the glycoprotein gB of Herpes simplex virus type 1 and bovine Herpes virus type 2 in infected cells. Arch Virol 108:1-17[Medline]
Roizman B, Sears AE (1990) Herpes simplex viruses and their replication. In Fields BN, Knipe DM, eds. Fields Virology. Vol 2. 2nd Ed. New York, Raven Press, 1795-1841
Stannard LM, Fuller AO, Spear PG (1987) Herpes simplex virus glycoproteins associated with different morphological entities projecting from the virion envelope. J Gen Virol 68:715-725[Abstract]
Torrisi MR, Lazzaro CD, Pavan A, Pereira L, CampadelliFiume G (1992) Herpes simplex virus envelopment and maturation studies by fracture label. J Virol 66:554-561[Abstract]
Van Bergen En Henegouwen PMP (1989) Immunogold labeling of ultrathin cryosections. In Hayat MA, ed. Colloidal Gold. Principles, Methods, and Applications. Vol 1. San Diego, Academic Press, 191-216
Vernon SK, Ponce de Leon M, Cohen GH, Eisenberg RJ, Rubin BA (1981) Morphological components of herpesvirus. III. Localization of Herpes simplex virus type 1 nucleocapsid polypeptides by immune electron microscopy. J Gen Virol 54:39-46[Abstract]
Ward PL, CampadelliFiume G, Avitabile E, Roizman B (1994) Localization and putative function of the UL20 membrane protein in cells infected with Herpes simplex virus 1. J Virol 68:7406-7417[Abstract]