![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Journal of Virology, March 2008, p. 3109-3124, Vol. 82, No. 6
0022-538X/08/$08.00+0 doi:10.1128/JVI.02124-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Eva Maria Borst,
Martin Messerle, and
Beate Sodeik*
Institute of Virology, Hannover Medical School, D-30625 Hannover, Germany
Received 26 September 2007/ Accepted 17 December 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HSV1 capsids can leave the nucleus by primary envelopment at the inner nuclear membrane (6, 64). According to the luminal or single-envelopment hypothesis, these enveloped virions present in the lumen of the nuclear envelope or the endoplasmic reticulum are further transported within the secretory pathway, and ultimately leave the infected cell upon fusion of a virion-containing vesicle or vacuole with the plasma membrane (12, 37, 55). Alternatively, according to the deenvelopment-reenvelopment hypothesis, the luminal virions shed their primary envelope by fusion with the outer nuclear membrane or membranes of the endoplasmic reticulum which is continuous with the nuclear envelope, resulting in cytosolic capsids (12, 37, 64). A third recent hypothesis suggested that nuclear capsids may also directly access the cytosol through dilated nuclear pores whose central channels with a regular width of about 50 nm may be widened sufficiently for direct egress of nuclear capsids (55, 94). Whatever their origin during egress, the cytosolic capsids can then be transported to the organelle of secondary envelopment, e.g., the trans-Golgi network (39, 89) or multivesicular bodies (11, 22). The resulting vesicle containing one or several virions then fuses with the plasma membrane, and releases virions into the extracellular medium (reviewed in references 12, 37, and 64).
To study the dynamics of entry, assembly or egress, viral particles can be labeled with fluorescent dyes or proteins to circumvent the limitations of immunolabeling. Since the direct attachment of fluorescence labels may alter the viral life cycle or the growth kinetics, electron as well as immunofluorescence microscopy are used to identify any potential differences between the wild type and the tagged strains (reviewed in references 31 and 78). There are several HSV1 strains in which fluorescent protein domains have been attached to structural proteins. In HSV1(KOS)-GFPVP26, the small capsid protein VP26 has been N-terminally labeled with green fluorescent protein (GFP) (29). The addition of GFP to VP26 interferes neither with cell entry and dynein-mediated microtubule transport to the nucleus (30) nor with dynein and dynactin binding to isolated capsids or capsid transport along microtubules in vitro (96). The addition of GFP to the envelope glycoprotein gB of HSV1 reduces plaque sizes by three- to fivefold and virus titers by 100-fold (70), whereas the addition of GFP or yellow fluorescent protein (YFP) to glycoprotein D (gD) seems to be better tolerated (66, 80).
One method to generate herpesvirus mutants relies on bacterial artificial chromosomes (BACs) as vectors to manipulate entire viral genomes in Escherichia coli (1). After bacterial mutagenesis in E. coli, such BAC-cloned viral genomes are transfected into eukaryotic cells, and the mutated viruses are recovered. Since the first cloning of a herpesvirus as a BAC (63), this technology has been used successfully to generate an ever growing number of viral mutants. The faithful construction and examination of viral mutants is facilitated, if the exact sequence of the targeted genome is known, as is the case for HSV1 strain 17+, which has been completely sequenced (60-62, 69) (GenBank accession no. NC_001806).
Here we describe a new BAC of HSV1(17+), in which the BAC sequences and a eukaryotic Cre recombinase expression cassette were flanked by loxP sites. We labeled the capsid protein VP26 and the envelope protein gD with fluorescent proteins to generate single-color and dual-color fluorescence viruses (cf. Table 1). Using these, we studied the subcellular itinerary of capsids and viral envelope proteins during the viral life cycle. Capsid egress from the nucleus commenced around 6 h postinfection (p.i.). The fluorescent viral capsids were followed through the cytosol to cytoplasmic membranes labeled by the fluorescent envelope protein, and dual-color fluorescence particles were secreted into the medium. Thus, the fluorescently labeled structural proteins faithfully reflected the intracellular trafficking of the wild-type HSV1 structural proteins during assembly and egress. Although some cells showed a dramatic reorganization of the nuclear pore network, changes in the nuclear pore architecture did not correlate with efficient nuclear capsid egress. Our data are most consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and fusion at the outer nuclear membrane to transfer capsids into the cytosol, followed by secondary envelopment of cytosolic capsids on cytoplasmic membranes.
|
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
VP26 (HSV1-K26Z [28]) and HSV1(KOS)-GFPVP26 (HSV1-K26GFP [29]), and the HSV1(F) was purchased from ATCC (VR-733; cf. Table 1). For single-step growth kinetics, BHK-21 cells were infected with a multiplicity of infection (MOI) of 5 PFU/cell for 48 h, and the resulting culture supernatants, as well as all virus preparations, were titrated on Vero cells (32). We used the rabbit polyclonal antibodies (PAb) 
-NC1 directed against VP5 (21), -VP26 against VP26 (28), R69 against gB (35), or R45 against gD (20), as well as the mouse monoclonal antibodies (MAb) 5C10 against VP5 (88), DL6 against gD (34), 7520 against gE (4), and LP11 against gH (9). Nuclear pore complexes were labeled with MAb 414 (Babco, Richmond, CA) (25).
Cloning of HSV1 as a BAC.
To insert the BAC sequences together with a β-galactosidase expression cassette and a single loxP site into the UL23 locus of HSV1, the recombination plasmid pblueLox-HomTK was constructed. The kanamycin resistance gene from pEYFP-ER (Clontech, San Jose, CA) was amplified with primers Kan-fwd and Kan-rev (cf. Table 2; all primers were purchased from MWG Biotech, Ebersberg, Germany), treated with NotI and XbaI, and cloned into pUC18NdeIlinker (84), resulting in pUC18NdeIlinker-Kan. DNA sequences homologous to 2 kb of UL23 and its flanking regions were amplified from HSV1(17+) DNA isolated from viral capsids (57). The 3' sequence (nucleotides [nt] 44591 to 46840) was amplified with 3'tk-fwd and 3'tk-rev, digested with XbaI and NheI, and ligated into pUC18Ndelinker-Kan cut with XbaI and NdeI. The 5' sequence (nt 47561 to 49773) was amplified with 5'tk-fwd and 5'tk-rev, digested with Bsp120I and NdeI, and ligated into the NotI- and NdeI-cut product of the previous cloning step, resulting in pHomTK. The homology cassette was released from pHomTK with SalI and cloned into the SalI site of pblueLox (79), yielding the recombination plasmid pblueLox-HomTK.
|
Insertion of a Cre recombinase gene. Two complementary oligonucleotides containing a loxP site flanked by NheI, PacI, XbaI, and NotI restriction sites (loxP-sense and loxP-antisense) were annealed and ligated into pUC18 digested with BamHI and HindIII, resulting in pUC18LoxP. A eukaryotic expression cassette for Cre recombinase with an intron was excised from pCreIn (79) with PacI and cloned into the PacI site of pUC18LoxP to yield pUC18LC. The tetracycline resistance cassette from pCP16 (16) was amplified with Tet-fwd and Tet-rev, cut with NheI, and cloned into the XbaI site of pUC18LoxP to construct pUC18LCT. 50-bp double-stranded DNA oligonucleotides homologous to sequences upstream of the BAC cassette were obtained by annealing complementary oligonucleotides (5'BAC-sense/5'BAC-antisense and 3'BAC-sense/3'BAC-antisense). These were ligated into the NheI and NotI sites of pUC18LCT, respectively, leading to pUC18LCTH. The complete 5.4-kb cassette was excised with NheI and NotI and inserted into the BAC pHSV1(17+)blue, resulting in pHSV1(17+)blueLox.
Reconstitution of HSV1(17+) strains from BACs. For transfection, BAC-DNA was prepared from 500-ml overnight E. coli cultures using the NucleoBond BAC 100 kit (Macherey & Nagel, Düren, Germany). A total of 5 x 105 Vero cells were grown overnight, transfected with 2 µg of BAC-DNA (MBS mammalian transfection kit), and cultured for several days until cytopathic effects developed.
Fluorescence tagging of the HSV1 proteins VP26 and gD.
VP26 was N terminally tagged with GFP by using pK26GFP (29). Sequencing of pK26GFP and of the DNA of HSV1(KOS)-GFPVP26 (29) revealed that in addition to the first four original codons of VP26, 50 bases in the noncoding region upstream of the VP26 open reading frame (ORF) were missing. The deleted region began 55 bp after the stop codon of UL34 (data not shown; P. Desai, unpublished data). For pK26RFP, the GFP sequence in pK26GFP was removed and replaced with the sequence of mRFP1 from pRSETB-mRFP1 (14). For pHSV1(17+)-BAC mutagenesis, an rpsLneo cassette was amplified from pRpsLneo (Gene Bridges GmbH, Dresden, Germany) with VP26rpsLneo-fwd and VP26rpsLneo-rev. This cassette replaced codons 4 to 7 of VP26 and interrupted its reading frame, generating pHSV1(17+)blueLox-VP26. The cassette was subsequently replaced with the PCR products obtained from plasmids pK26GFP or pK26RFP using the primers VP26Hom-fwd and VP26Hom-rev. The resulting BACs were named pHSV1(17+)blueLox-GFPVP26 or -RFPVP26.
gD was tagged with GFP or mRFP1 using an en-passant mutagenesis technique (87). The GFP insertion construct was amplified from pEP-EGFP-in (81) with the primers gDGFP-fwd and gDGFP-rev and introduced into pHSV1(17+)-BACs. The mRFP1 sequence was amplified from pEP-mRFP1-in (81) with gDRFP-fwd and gDRFP-rev. Positive clones were transformed with the plasmid pBAD-I-SceI (87). I-SceI expression was induced by L-arabinose, and Red enzyme expression was induced by heat shock. The bacteria were grown with chloramphenicol, ampicillin, and L-arabinose, and kanamycin-sensitive clones were screened by restriction analysis.
DNA electrophoresis and Southern blotting. DNA fragments were separated in agarose gels, blotted onto nylon membranes, hybridized, and probed with digoxigenin (DIG)-labeled DNA probes (Roche, Mannheim, Germany). A probe against BAC sequences was generated by PCR with primers BAC-fwd and BAC-rev using the PCR DIG probe synthesis kit. A probe against the HSV1 replication origin oriL was produced with oriL-fwd and oriL-rev.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Protein samples were separated on linear 5 to 15% polyacrylamide gradient gels (52). After transfer onto nitrocellulose membranes, the samples were incubated with primary antibodies, followed by secondary antibodies coupled to alkaline phosphatase (Dianova, Hamburg, Germany) and developed with BCIP (5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazolium salt.
Light microscopy. Vero cells grown on coverslips were infected with HSV1 at an MOI of 10 PFU/cell. In some experiments (Fig. 7 and 8), the cells were synchronously infected by inoculation with HSV1 for 2 h on ice in a small volume and then further incubated at 37°C (32, 81). After 1 h, the cells were washed with 40 mM citric acid-135 mM NaCl-10 mM KCl (pH 3) to inactivate extracellular virions (38, 95). Fresh medium was added, and at different time points the cells were fixed and immunofluorescence labeling was performed as described previously (81). The HSV1 Fc receptor (33) was blocked by using 10% (vol/vol) human serum of healthy, HSV1-seronegative volunteers (30). Secondary antibodies were coupled to lissamine-rhodamine sulfonyl chloride, fluorescein isothiocyanate (FITC), or Cy5 (Dianova). The specimens were analyzed on a Axiovert 200M microscope equipped with an LSM 510 META confocal laser scanning unit (Zeiss, Jena, Germany) using a Plan-Apochromatic x63 oil immersion objective lens with a 1.4 numeric aperture. For a quantitative analysis of the HSV1 egress pathway, at least 85 randomly chosen cells were scored for several time points from 4 to 12 h p.i. Image acquisition and processing was performed by using the Zeiss LSM imaging software, ImageJ 1.35 (Wayne Rasband; National Institute of Health [http://rsb.info.nih.gov/ij/]), and Adobe Photoshop CS (Adobe Systems, San Jose, CA).
|
|
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
The pHSV1(17+)blue clones were analyzed by restriction enzyme digestion. Expected fragment sizes were calculated according to the published HSV1(17+) sequence (GenBank accession no. NC_001806). Novel fragments resulting from the BAC insertion were detected after NotI and EcoRI digestion (Fig. 1B and asterisks in Fig. 1C and D, lanes B1, B2, and B3). For the viral DNA (Fig. 1C, lane 17+), the genome termini and the NotI joint fragment were not recovered as distinct bands. Instead, there was a very weak, diffuse signal at around 3 kb (not shown) due to the varying numbers of the repetitive 400-bp a-sequence in HSV1 (27, 74). Furthermore, the a-sequence itself can contain various numbers of direct repeats (27). In pHSV1(17+)blue, the two joint fragments had definite sizes that varied between clones (Fig. 1C, arrowheads in lanes B1, B2, and B3). According to the published sequence, both NotI joint fragments have a size of 2,918 bp, as in one clone (Fig. 1C, lane B3), whereas other clones (Fig. 1C, lanes B1 and B2) showed permutations of the joint fragment. Thus, the joint fragments were fixed as definite structures in the BACs recovered from E. coli but varied in length in the HSV1 genomes isolated from eukaryotic cells. Due to the circular organization, the 3'-terminal EcoRI fragment of linear HSV1-DNA forming a diffuse band around 5.6 kb (Fig. 1D, arrowhead in lane 17+) was absent in the BAC-DNAs. In all clones, the 2.0-kb BamHI fragment spanning the transition between the US region and the adjoining inverted repeat was reduced by about 50 bp (not shown). Variations in a highly repetitive sequence in this noncoding region have been observed before for individual HSV1 isolates (90).
All three pHSV1(17+)blue clones were infectious after transfection into Vero cells, and clone B1 was chosen for further analysis and mutagenesis.
Stability of pHSV(17+)-BACs. Since repetitive regions could provide targets for unwanted recombination in bacteria, the stability of the HSV1-BACs was tested. After 20 passages of pHSV1(17+)blue in E. coli, no major changes were observed in the restriction fragment patterns using six different enzymes (Fig. 2A). However, one of the NotI joint fragments had decreased in size (Fig. 2A, arrowhead), suggesting that a deletion had occurred in the highly repetitive a-sequences. Moreover, an EcoRV fragment also slightly shifted to a lower size (Fig. 2A, asterisk), probably by shortening of a repetitive sequence in a noncoding region upstream of UL1 (see also reference 90). However, BAC DNA prepared from both passages 1 and 20 induced cytopathic effects with comparable kinetics after transfection into Vero cells (data not shown). The HSV1 replication origin oriL consists of a 144-bp palindrome which, in addition, contains several direct repeats (47). All of the HSV1-BACs we tested, including a BAC of HSV1(F) (86), had lost their oriL (Fig. 2B). In contrast, a PCR revealed no changes in oriS between the HSV1(17+) wild type and the corresponding BACs (data not shown). Thus, with the exception of the indicated changes, the BAC pHSV1(17+)blue and its derivatives were stably maintained in E. coli and retained their infectivity upon transfection into eukaryotic cells.
|
Compared to HSV1(17+), the BAC-derived virus HSV1(17+)blue exhibited a moderate growth defect (Fig. 3A). HSV1(17+)blueLox was slightly less attenuated than HSV1(17+)blue, with its titers being reduced 
5-fold compared to HSV1(17+) (Fig. 3A). Thus, HSV1(17+)blueLox was attenuated compared to the wild type, but the excision of the BAC sequences was beneficial. Similar to HSV1(17+)blueLox, the mutant HSV1(KOS)-tk12 (92) also contains a β-galactosidase expression cassette instead of UL23, but HSV1(KOS)-tk12 showed no growth defect in BHK cells compared to HSV1(KOS) (Fig. 3B).
|
VP26, -GFPVP26, and -RFPVP26 showed no alterations except the desired modifications and, again, permutations in the NotI joint fragments (Fig. 1C and D, lanes , G, and R). HSV1(17+)blueLox-GFPVP26 and -RFPVP26 secreted less infectious virus compared to HSV1(17+)blueLox but produced higher titers than HSV1(17+)blueLox-VP26 (Fig. 3C). Similarly, HSV1(KOS)-GFPVP26 produced lower titers than HSV1(KOS), but there was no growth difference between HSV1(KOS)-VP26 and HSV1(KOS)-GFPVP26 (Fig. 3D). Vero cells were then infected with HSV1(17+)blueLox-GFPVP26 (Fig. 4A to D) or -RFPVP26 (data not shown), fixed, and labeled with anti-VP26 and with anti-VP5, directed against the major capsid protein. At 9 h p.i., GFPVP26 (Fig. 4A) was mostly localized in the nucleus, either on single capsids or on capsid aggregates, but there were also many capsids in the cytoplasm. The GFPVP26 signal (Fig. 4A and green area in Fig. 4D) colocalized with the anti-VP26 labeling (Fig. 4B and red area in Fig. 4D) on single capsids and nuclear capsid aggregates (yellow area in Fig. 4D). VP5 was mostly detected on nuclear capsids and aggregates (Fig. 4C and blue area in Fig. 4D), where it colocalized with GFPVP26 and anti-VP26 (white area in Fig. 4D). Some GFPVP26-containing nuclear capsids were stronger labeled by anti-VP5 (blue area in Fig. 4D) than other nuclear and cytoplasmic capsids (green and yellow areas in Fig. 4D). Similar results were obtained with HSV1(17+)blueLox-RFPVP26 (data not shown). Thus, GFPVP26 and RFPVP26 were recruited onto newly synthesized nuclear capsids. Cytoplasmic and nuclear capsids contained comparable amounts of GFPVP26 or RFPVP26, since the intensity of their fluorescence was rather homogeneous. In contrast, the antibodies to VP26 and VP5 had less access to the surface of cytoplasmic capsids, probably due to tegumentation and envelopment.
|
|
Since the detection of GFP is more sensitive than that of RFP, we also analyzed HSV1-(17+)-RFPVP26-gDGFP (Fig. 4I to L). At 9 h p.i., there were numerous cytoplasmic capsids of which a large portion did not colocalize with either glycoprotein and thus most likely represented unenveloped, naked cytosolic capsids (open arrowhead and red area in Fig. 4L). There were also cytoplasmic membranes which contained gDGFP (green area in Fig. 4L) and/or gB (blue area in Fig. 4L; colocalization turquoise in Fig. 4L) but no capsids. Other capsids colocalized with gDGFP and gB (filled arrowhead and white area in Fig. 4L), suggesting that RFPVP26, gDGFP and gB (as well as GFPVP26 and gDRFP) had been correctly targeted to the cytoplasmic site of HSV1 assembly.
Dual-color fluorescence HSV1 virions.
We next analyzed the protein composition of viral particles secreted from infected cells (Fig. 6A). An anti-VP26 antibody detected a single band at the expected molecular weight in wild-type and HSV1(17+)blueLox but no signal in HSV1(17+)blueLox-VP26. The bands at around 40 kDa represented the fluorescent VP26 fusion proteins, which were incorporated in full length into HSV1 particles. Probing with an anti-gD antibody showed an increase in the molecular weight of gD, indicating the incorporation of the full-length fusion protein into virions. A fraction of gDRFP was most likely cleaved at the gD-RFP-junction, since we still observed a minor signal at the original gD position (Fig. 6A, lane GR). Similar results were obtained for HSV1(17+)blueLox-RFPVP26-gDGFP (data not shown).
|
Time course of HSV1 nuclear egress and assembly. The nuclear pores are the gateways of the nuclear envelope that control macromolecular transport between the cytosol and the nucleoplasm. Recent studies suggested that nuclear pore complexes can become dilated during a herpesvirus infection and that HSV1 capsids with their diameter of 125 nm might also directly traverse through such pores to the cytosol (55, 65, 94). To obtain an overview of the HSV1 induced changes in the nuclear pore network, we infected Vero cells with 10 PFU/cell of either HSV1(17+)blueLox or of HSV1(17+)blueLox-GFPVP26-gDRFP and analyzed them from 2.5 to 12 h p.i. For the first, the small capsid protein VP26 was labeled with specific antibodies (data not shown [but cf. Fig. 4 and 8]), whereas for the latter VP26 (Fig. 7A to E) and gD (Fig. 7F to J) were detected by their intrinsic fluorescence. The nuclear pores were labeled with MAb 414 (25), which recognizes phenylalanine-glycine (FG) repeats present in several proteins of the nuclear pore complexes (26) (Fig. 7K to O).
At 2.5 h (results not shown) and at 4 h p.i., the cells contained almost no nuclear capsids, but some incoming capsids derived from the inoculum were still located on the nuclear rim (Fig. 7A and green area in Fig. 7P) (cf. references 31, 32, and 81). As the infection progressed, many cells contained increasing numbers of nuclear capsids, and capsid aggregates developed in more and more nuclei (cf. Fig. 7C). At 8 h p.i., and more prominently at 10 h p.i., newly synthesized capsids had reached the cytoplasm (arrowheads in Fig. 7R and S) and later often accumulated there in clusters (Fig. 7E and T). Newly synthesized gDRFP was detected as early as 4 h p.i. (Fig. 7F). From 6 to 12 h p.i., more and more gD was concentrated in cytoplasmic clusters (Fig. 7G and J). At 10 h p.i. and more so at 12 h p.i., the cytoplasmic capsids colocalized with gD in these clusters (yellow areas in Fig. 7S and T).
Labeling of the DNA (data not shown) or the nuclear pores enabled a distinction of nuclear from cytoplasmic capsids by confocal fluorescence microscopy. Nuclear capsids rarely colocalized with gD and, if so, only did so in the nuclear periphery (results not shown). Several cytoplasmic capsids did not colocalize with gD (arrowheads in Fig. 7R, S, and T), but as the infection progressed many had translocated to those regions of the cytoplasm that also contained a high concentration of gD (Fig. 7T and cf. Fig. 4L). According to the luminal pathway hypothesis (12, 37, 55), HSV1 capsids acquire their final envelope at the inner nuclear membrane, and one would expect a stronger colocalization of GFPVP26 with gDRFP at the nuclear envelope and throughout the entire cytoplasm. However, our data were most consistent with the deenvelopment-reenvelopment hypothesis of HSV1 assembly (12, 37, 64), which predicts that initially cytosolic capsids do not colocalize with gDRFP, but upon secondary envelopment in the cytoplasm acquire their secondary, final envelope containing gDRFP.
Changes in the nuclear pore network during HSV1 infection. The nuclear pores in mock-infected cells were mostly randomly distributed over the entire nuclear surface, yielding a ring-like, rim appearance of the nuclear pore network around the nucleus in confocal images (Fig. 8A). Moreover, several cytoplasmic spots contained a high concentration of nuclear pore antigens (open arrowhead in Fig. 8A). Some cells showed a particularly weak labeling, and others showed a particularly strong labeling, which most likely reflected cell-cycle-dependent changes in the concentration of nuclear pores and in the organization of the nuclear pore had (10, 58). At 8 h p.i., the morphology of the nuclear pore network had changed in some cells. Occasionally, the nuclear envelope appeared to be extended or invaginated into the nuclear interior, as suggested by a dotted line appearance of nuclear pore complexes within the boundaries of the ring-like labeling of the outer nuclear diameter (Fig. 8A, "fold"). In some cells, the labeling of the ring-like and the apparently invaginated nuclear envelopes was interrupted by large gaps (Fig. 8A, "irregular"). The most striking phenotype displayed nuclei with a ring-like labeling of rather low intensity, and instead the nuclear pores had been clustered on the nuclear envelope (Fig. 8A, "cluster," filled arrowhead).
Since the morphology of the nuclear pore network was heterogeneous after HSV1 infection, we randomly sampled cells for several time points p.i. and sorted them, depending on their nuclear pore labeling, into five different classes: weak or no label, regular, fold, irregular, and cluster (Fig. 8A). For the mock-infected cells, almost 90% of the nuclei were characterized by a "regular" morphology (Fig. 8B). At 8 h p.i., ca. 20 to 25% of the cells had acquired the described changes in the nuclear pore network (fold, irregular, and cluster). The fraction of cells characterized by changes in the nuclear pore architecture then did not change further up to 12 h p.i. The different distribution of the nuclear pore phenotypes in the uninfected, mock-treated cells reflected the heterogeneity in the nuclear pore architecture among different cell populations (compare HSV1(17+)blueLox to HSV1(17+)blueLox-GFPVP26-gDRFP in Fig. 8B).
For all time points, the infected cells were also categorized into four classes according to the stage of the HSV1 infection as judged by the amount of cytoplasmic capsids (Fig. 8C). At 4 h p.i., almost 90% of the cells infected with HSV1(17+)blueLox and ca. 40% of those infected with HSV1(17+)blueLox-GFPVP26-gDRFP contained fewer than 10 cytoplasmic capsids but no nuclear capsids (cf. Fig. 7A and P), suggesting that most if not all of these cytoplasmic capsids were derived from the inoculum (30, 81). At 8 h p.i., ca. 25 to 50% of the cells contained more than 10 cytoplasmic capsids. This proportion increased at later time points. However, particularly at 12 h p.i., many cells had been lost due to cytopathic effects and detachment. Overall, more cytosolic capsids of HSV1(17+)blueLox than of HSV1(17+)blueLox-GFPVP26-gDRFP were detected, which most likely reflected that the infection with the dual-color fluorescence HSV1 strain progressed slower (cf. Fig. 5C).
Intriguingly, the number of cells characterized by changes in the nuclear pore network increased (Fig. 8B) with kinetics similar to those of the accumulation of cytoplasmic capsids (Fig. 8C). We therefore sought to determine whether, independent of the time point, reorganization of the nuclear pore network correlated with the amount of capsids that had egressed from the nucleus to the cytoplasm. All cells recorded from 6 to 12 h p.i. were therefore sorted into the four classes depending on the concentration of cytoplasmic capsids (none, fewer than 10, 10 to 50, and more than 50), and further into the five classes depending on the morphology of the nuclear pore network but irrespective of the time point postinfection analyzed (Fig. 8D). Of the cells containing a high concentration of cytoplasmic capsids (i.e., more than 10), 80 or 60% had an apparently regular nuclear pore network after infection with HSV1(17+)blueLox or HSV1(17+)blueLox-GFPVP26-gDRFP, respectively. However, ca. 20 to 25% of all cells showing no cytoplasmic capsids were characterized by HSV1 induced changes in the architecture of the nuclear pore network (fold, irregular, and cluster). This proportion remained rather constant for HSV1(17+)blueLox and increased up to ca. 40% for HSV1(17+)blueLox-GFPVP26-gDRFP as the concentration of cytoplasmic capsids increased to more than 50 per cell. The amount of cytoplasmic capsids was not higher in cells in which the nuclear pore architecture was not regular. In another experiment with a slower infection at a lower MOI, 50 to 75% of the cells with a high amount of cytoplasmic capsids showed major changes in the nuclear pore architecture (data not shown). Thus, surprisingly the organization of the nuclear pore network was less heterogeneous among cells infected with a higher MOI.
In summary, the percentage of cells characterized by HSV1 induced alterations in the nuclear pore architecture increased around 8 h p.i. irrespective of whether VP26 and gD were tagged or not. Nevertheless, irrespective of the virus dose or strain, the HSV1-induced changes in the nuclear pore network were not a prerequisite for HSV1 nuclear capsid egress, since many cytoplasmic capsids were present in cells with an apparently regular nuclear pore distribution.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All HSV1-BAC clones we analyzed lacked the 144-bp oriL, which displays a notorious instability upon cloning (93). This palindromic sequence has only been maintained in an E. coli strain that lacks several DNA repair and recombination genes and thus increases the stability of inverted repeats and palindromes (47). However, such a strain is not suitable for BAC mutagenesis, since the oriL most likely would become unstable as soon as recombination enzymes required for mutagenesis would be expressed. Since oriL is not required for replication in cell culture, its absence will not hamper most experiments. However, deleting the oriL results in reduced mortality and pathogenicity in a corneal mouse in vivo infection model (7). For such studies, it may become necessary to construct HSV1 mutants by combining BAC mutagenesis with recombination in eukaryotic cells. Mutagenesis may be performed in an HSV1 BAC lacking oriL and one of its neighboring essential genes. The respective DNA fragment harboring the lacking elements may be maintained in the special E.coli strain and, after cotransfection with the mutated BAC into eukaryotic cells, replication-competent HSV1 mutants with intact oriL may be generated.
The expression of Cre recombinase led to the deletion of the BAC sequences after transfection into eukaryotic cells and faster replication, as reported for pseudorabies virus, human cytomegalovirus, and murine gammaherpesvirus 68 (2, 79, 97). The lower infectivity of HSV1(17+)blueLox compared to HSV1(17+) was probably neither due to the insertion of a β-galactosidase expression cassette nor to the deletion of UL23 but might have been caused by the deletion of oriL. We cannot exclude that there were additional differences between the HSV1(17+)-BACs and HSV1(17+). HSV1(strain F)-BAC viruses are not attenuated compared to the wild type (86). Interestingly, already the mere addition of a single loxP site into HSV1(17+) can result in a threefold titer reduction (43). Despite its slight attenuation, we considered the BAC pHSV1(17+)blueLox a useful base for the construction of dual-fluorescence strains.
Dual-color fluorescence HSV1(17+). HSV1(KOS)-GFPVP26, in which GFP has been added to VP26 (29), has the same cell entry characteristics as HSV1(KOS) (30). Sufficient amounts of GFP-labeled VP26 remained bound to capsids, so that their subcellular localization and motility could be analyzed in living cells (J. Janus, K. Döhner, A. Wolfstein, F. Büttner, S. Schmidt, and B. Sodeik, unpublished data). Moreover, GFPVP26-labeled capsids were used to study microtubule mediated transport of HSV1 capsids in vitro (96). Using antibodies specific to VP26 and VP5, we confirmed that the GFPVP26 and RFPVP26 signals represented HSV1 capsids (30; the present study) and that the fusion proteins gDRFP and gDGFP colocalized with a gD labeling, as recently also described for gDYFP (80). Adding GFP to gD reduced the amount of infectious virus secreted from BHK-21 cells. The addition of YFP to the cytosolic tail of gD was less detrimental to virus growth in Vero cells, whereas, after amplification in a neuroblastoma cell line, the titers are decreased (80). Thus, manipulating gD has different effects in different cell lines. The cytoplasmic tail of gD is dispensable for growth in cell culture (41), but adding GFP or RFP to it may reduce its affinity to tegument or envelope proteins (17, 40). However, ultimately HSV1 particles that incorporated both fluorescent fusion proteins and contained several other envelope proteins were secreted. Thus, the trafficking of the fluorescence-labeled gD was similar to untagged HSV1 glycoproteins. These dual-color fluorescence HSV1 strains provide promising tools for the analysis of assembly and egress (the present study), as well as for the study of cell entry (K. Döhner, C.-H. Nagel, and B. Sodeik, unpublished data). Moreover, these strains allow the detection of all subviral particles containing tagged VP26 and gD, irrespective of their interaction and possible masking by host factors or other HSV1 structural proteins.
Nuclear pore network during HSV1 nuclear capsid egress and assembly. During HSV1 infection, many host organelles and also the cytoskeleton change their morphology and subcellular organization. These HSV1-induced changes are collectively summarized under the term cytopathic effects. For example, the membranes containing antigens of the Golgi apparatus are scattered over the entire cytoplasm rather than being localized in a tight juxtanuclear cluster, and the microtubules are marginalized to the cell periphery (5, 36, 51). Moreover, the nuclear envelope is severely modified during HSV1 infection (8, 46, 53, 77), resulting also in a reorganization of the nuclear pore network (55, 71). Together with the recent electron microscopy observations that some nuclear pores appear to become dilated during infection with alphaherpesviruses, this has led to the hypothesis that HSV1 induces changes in the nuclear pore network, and the widening of the lumen of individual nuclear pores might generate a channel large enough to allow a passage of nuclear capsids directly into the cytosol (55, 94). In this scenario, the capsids would step aside and bypass the barriers posed by the nuclear lamina and the inner and outer nuclear membranes.
Simultaneous recording of the subcellular localization of nuclear pore complexes, capsids, and envelope proteins allowed us to distinguish both nuclear from cytoplasmic capsids and virions in vesicles from cytosolic capsids. In accordance with Leuzinger et al. (55), the nuclear pore network was modified as the HSV1 infection progressed. However, in the majority of cells containing many cytoplasmic capsids, the nuclear pore architecture was not changed, suggesting that changes in the nuclear pore architecture were not required for efficient nuclear egress. Our data are consistent with studies using HSV1 deletion mutants. Without US3 (72) or the glycoproteins gB and gH (40) capsids can acquire a primary envelope at the inner nuclear membrane but are stuck in the perinuclear space between inner and outer nuclear membrane, presumably due to an inability to catalyze primary fusion. In the absence of UL31 (15) or UL34 (75), capsids are defective in primary budding, and nuclear capsids accumulate but are also unable to use potentially dilated nuclear pores as gateways to the cytosol. Leuzinger et al. (55) proposed that the changes in the nuclear pore labeling observed by fluorescence microscopy may reflect an extension of the luminal channel of individual nuclear pores as detected by electron microscopy. However, the apparent holes in the nuclear envelope may not contain bona fide nuclear pore proteins at their rims. If they would indeed lack nuclear pore proteins, they may not have arisen from dilation of preexisting pore complexes but might have been generated by HSV1-catalyzed fusion of the inner with the outer nuclear membrane. Moreover, the nuclei with irregular or clustered nuclear pore architectures might have clustered several nuclear pore complexes within a small area of the nuclear envelope rather than indeed enlarged the nuclear pore channel. The latter scenario agrees well with many electron microscopy studies that reported that the integrity of individual nuclear pores appeared intact until late in herpesvirus infection (40, 46, 64, 65, 72).
Our data are consistent with nuclear capsids leaving the nucleus via primary budding and primary fusion to release capsids into the cytosol rather than with a direct passage through a dilated nuclear pore. In addition, only later in infection gD had accumulated in the membranes surrounding the nucleus, whereas gD was rather prominent on cytoplasmic membranes, where it often colocalized with capsids. The cytoplasmic capsids that did not colocalize with gD and gB most likely represented cytosolic capsids rather than capsids inside virions. The resulting cytosolic capsids would then be transported to cytoplasmic membranes to acquire a final envelope by secondary envelopment. Our results are expected for capsids that follow the deenvelopment-reenvelopment pathway, whereas the luminal pathway for HSV1 egress predicts that capsids should already acquire gD and gB at the inner nuclear membrane and that all cytoplasmic capsids colocalize with all viral membrane proteins.
These dual-color fluorescence strains will be valuable tools for evaluating the fate of other host organelles and the cytoskeleton during the viral life cycle, for studying the dynamics and sequence of events in HSV1 assembly and entry in living cells, and for further characterizing the phenotypes of mutations in HSV1 structural proteins. Such studies will result in further insights into the HSV1-induced cytopathic effects and whether these are kinetically linked to HSV1 nuclear capsid egress, assembly, and secretion of infectious particles or whether they constitute bona fide bystander effects of viral infection.
|
ACKNOWLEDGMENTS |
|---|
This study was supported by the German Research Council (DFG; So403/2, So403/3 to B.S.), by the EU 6th Framework Program (EU-NEST AXON SUPPORT, contract number 12702 to B.S.), and by the Wilhelm-Sander-Foundation (grant 2004.075.1 to M.M.). C.-H.N. received a fellowship from the Hannover Biomedical Research School (DFG-GK745 on Mucosal Host-Pathogen Interaction).
|
FOOTNOTES |
|---|
Published ahead of print on 26 December 2007.
Present address: CSIRO, Canberra, Australia.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
