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Journal of Virology, October 2008, p. 10218-10230, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00859-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Efficient Quiescent Infection of Normal Human Diploid Fibroblasts with Wild-Type Herpes Simplex Virus Type 1{triangledown}

Robert McMahon and Derek Walsh*

National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland

Received 23 April 2008/ Accepted 3 August 2008


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ABSTRACT
 
Quiescent infection of cultured cells with herpes simplex virus type 1 (HSV-1) provides an important, amenable means of studying the molecular mechanics of a nonproductive state that mimics key aspects of in vivo latency. To date, establishing high-multiplicity nonproductive infection of human cells with wild-type HSV-1 has proven challenging. Here, we describe simple culture conditions that established a cell state in normal human diploid fibroblasts that supported efficient quiescent infection using wild-type virus and exhibited many important properties of the in vivo latent state. Despite the efficient production of immediate early (IE) proteins ICP4 and ICP22, the latter remained unprocessed, and viral late gene products were only transiently and inefficiently produced. This low level of virus activity in cultures was rapidly suppressed as the nonproductive state was established. Entry into quiescence was associated with inefficient production of the viral trans-activating protein ICP0, and the accumulation of enlarged nuclear PML structures normally dispersed during productive infection. Lytic replication was rapidly and efficiently restored by exogenous expression of HSV-1 ICP0. These findings are in agreement with previous models in which quiescence was established with HSV mutants disrupted in their expression of IE gene products that included ICP0 and, importantly, provide a means to study cellular mechanisms that repress wild-type viral functions to prevent productive replication. We discuss this model in relation to existing systems and its potential as a simple tool to study the molecular mechanisms of quiescent infection in human cells using wild-type HSV-1.


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INTRODUCTION
 
Herpes simplex virus-1 (HSV-1) is a large, double-stranded DNA virus that establishes life-long latent infection in sensory neurons of affected individuals after spread at the primary site of infection (59). Lytic replication of the virus is well characterized and involves the temporally ordered expression of viral genes, broadly grouped into three classes, termed immediate-early, early, and late. The outcome of lytic replication is death of the host cell. However, during primary infection neurons that do not support lytic replication ultimately play host to the viral genome in a nonproductive state, termed latency, in which minimal transcription from the viral genome occurs. In response to certain environmental cues, such as UV light and stress, HSV-1 periodically reactivates from latency, replicating in ganglia and the differentiated epithelia that they innervate.

The complex nature of latent infection means that it has proven challenging to study at the cellular level. Latency can be experimentally reproduced in animals, most commonly using rodents or rabbits, and these models have been used to develop a broad understanding of factors that regulate various aspects of the latent state (59). However, the inherent complexity of animal models means that they are largely unsuited to detailed mechanistic studies. To elucidate latency at the cellular level, a number of in vitro models have been developed in which the virus enters a nonproductive or quiescent state that mimics the primary features of in vivo latency. Cell types used to establish such models have included primary cultures of explanted ganglia (78, 83-87), T-lymphoid cells (27, 89), and hyperresistant neuronal cell lines (39, 41, 52, 73) as well as three-dimensional keratinocyte cultures (72). HSV quiescence has also been extensively studied in nerve growth factor-differentiated PC12 cells, a rat neuronal cell line (4, 12-15, 46, 51, 71),

Ethical and accessibility issues limit the development of human neuronal systems using primary cell cultures (79). Therefore, nonproductive infection of human cells has been studied most extensively in cultures of primary fibroblasts. The ability of human fibroblasts to support quiescent infection is thought to be due to their low metabolic state, which may resemble that of neurons more closely than transformed cell lines, or their expression of factors that restrict virus replication (28). Fibroblast models almost invariably exploit important early findings that demonstrated the sensitivity of HSV replication to temperature elevation (11, 32, 77). Experiments by Crouch and Rapp (10) showed that replication of HSV was severely suppressed in certain cell types by elevating the temperature to 40.5°C as long as the infection had not progressed for more than 4 to 6 h. This demonstrated that replication of the virus itself was not sensitive to temperature elevation but that an early stage in the virus life cycle was inhibited in a host cell-specific manner. Initially, quiescence was established in fibroblast cultures with the combined use of temperature elevation and chemical inhibitors of virus replication (9, 53, 55, 65, 67, 80-82). Quiescent infection with wild-type HSV-2 was subsequently achieved in the absence of inhibitors solely by using temperature elevation to 42°C in human embryonic lung cells but only to multiplicities of around 0.003 PFU/cell (29, 60, 90). Due to the reported cytotoxicity of immediate early (IE) gene expression by wild-type virus in such cell types (38), efficient quiescent infection on a population-wide scale was achieved and more extensively studied by infecting human fibroblast lines with viral mutants that are deficient in various IE genes (18, 21, 30, 31, 36, 57, 58, 61, 63, 69), often employing elevated temperatures or chemical inhibitors to prevent low-level replication of these mutants. The ability of these mutants to establish latent infection in animal models (8, 43, 68, 74) validated the findings made with these in vitro systems and has led to the suggestion that few, if any, viral gene products are actually required to establish a latent or nonproductive state.

However, whether viral gene expression occurs during entry into the latent state remains unclear. While viral gene expression may not be required to establish quiescence, it has been reported during latency in vivo and quiescence in vitro (1, 17, 18, 40, 66, 74), suggesting that neurons surviving primary infection may tolerate at least some level of virus activity while repressing lytic replication resulting in a latent infection. Understanding how cells that support nonproductive infection tolerate and suppress viral protein production or function will be important to understanding processes governing the natural host-pathogen balance that determines whether lytic or latent infection ensues. Here, we show that when primary normal human diploid fibroblasts were serum starved and incubated at elevated temperatures prior to infection to induce the expression of heat shock proteins (HSPs), known to reduce cellular stress, efficient quiescent infection was established with wild-type HSV-1 without the use of chemical inhibitors of virus replication. The characteristics of this model bore striking similarities to existing in vivo and in vitro systems and demonstrated that although some IE gene products were made efficiently during entry into quiescence, these cells suppressed the accumulation of ICP0 and failed to support productive infection. This simple and efficient model should provide a useful means of studying the cellular mechanisms involved in host repression of lytic replication and the establishment of a quiescent state using wild-type HSV-1.


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MATERIALS AND METHODS
 
Cells, viruses, and conditions to establish quiescence. Normal human diploid fibroblasts (NHDFs) (Clonetics) and Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum ([FBS] catalog no. F7524; Sigma,), 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 units of penicillin and 50 µg of streptomycin per ml. HSV-1 strains KOS and Patton were a gift of Ian Mohr, New York University School of Medicine. Virus was propagated, and titers were determined on Vero cells as described previously (76). Adenoviral vectors, a gift of Saul Silverstein, Columbia University, were propagated, and titers were determined on low-passage 293 cells as previously described (90). To establish quiescent infection, confluent wells of six-well plates or 35-mm dishes of low-passage NHDFs were serum starved by washing three times in 2 ml of phosphate-buffered saline (PBS) and cultured for 3 days in 1.5 ml of DMEM containing 0.2% FBS. Cultures were then transferred to a CO2 incubator at a temperature of 37°C or 41°C, as indicated on the figures, for a further 24 to 30 h and then infected at a multiplicity of infection of 0.5 to 1 PFU/cell in a volume of 1 ml without rocking to avoid temperature fluctuations. After 3 h virus was removed, and cells were washed, refed with temperature-equilibrated 0.2% FBS-DMEM, and then returned to the appropriate temperature. For reactivation experiments, cultures were infected at a multiplicity of 15 PFU per cell with an adenoviral vector or medium alone, as indicated in the figure legends, in a volume of 500 µl at 37°C. Cultures were rocked every 20 min for 2 h; the medium was then removed, and cells were washed in 5% FBS-DMEM and maintained in 2 ml 5% FBS-DMEM. In the case of experiments using human serum ([HS] catalog no. P2918; Sigma) to prevent secondary spread of virus, after removal of the adenovirus and the washing step, cultures were maintained in 2 ml of 5% HS-DMEM.

Antibodies and chemicals. Antibodies toward ICP0 (catalog no. Ab6513), ICP4 (catalog no. Ab6514), ICP5 (catalog no. Ab6508), and PML (promyelocytic leukemia protein) (catalog no. Ab53773) were obtained from Abcam, United Kingdom. Antibodies toward Hsp27 (catalog no. 2402), Hsp70 (catalog no. 4873), poly(A) binding protein ([PABP] catalog no. 4992), and p38 (catalog no. 9212) were obtained from Cell Signaling Technologies (United States). Anti-Us11 and anti-ICP22 antisera were gifts of Richard Roller, University of Iowa, and John Blaho, Mount Sinai School of Medicine, respectively.

Metabolic labeling, Western blotting, and immunofluorescence. For each well of six-well plates, medium was changed to 1 ml of DMEM without methionine or cysteine (catalog no. D0422; Sigma-Aldrich) containing HEPES, pH 8, sodium pyruvate, L-glutamine, penicillin-streptomycin and 77 µCi of [35S]methionine-cysteine (catalog no. NEG072; Perkin Elmer) for 1 h, as previously described (76). Total cellular protein was solubilized in 250 µl of sample buffer and boiled for 3 min. Samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then fixed gels were dried under vacuum for 2 h at 80°C and exposed to X-ray film. For Western blotting, samples were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with antiserum, as indicated on the figures. After a washing step, blots were probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (catalog no. 31430 and 31460; Pierce), processed, and subjected to chemiluminescence detection according to the manufacturer's instructions (catalog no. 32106; Pierce). For immunofluorescence cells were seeded on 35-mm dishes or wells of six-well plates containing glass coverslips and infected as described above. At the point of harvesting, cells were washed in PBS, fixed for 15 min in 4% paraformaldehyde, and then extensively washed again in PBS. Cells were permeabilized in 0.1% Triton X-100 Tris-buffered saline for 30 min and then washed and blocked for a further 30 min at room temperature. Samples were incubated with a primary antibody, as indicated on the figures, in Tris-buffered saline-0.1% Tween for 1 h at room temperature and then washed and incubated with the appropriate fluorescently labeled secondary antibody for 40 min, as indicated in the figure legends. Cross-adsorbed secondary antibodies were fluorescein isothiocyanate (FITC)-labeled anti-mouse (catalog no. F0261) from Dako or tetramethyl rhodamine isothiocyanate-labeled anti-mouse (catalog no. 715-025-150) and FITC-labeled anti-rabbit (catalog no. 711-095-152) from Jackson ImmunoResearch, United Kingdom. Nuclei were counterstained with Hoechst. Images were captured on a Leica DFC 500 microscope at the magnification indicated in the figure legends.


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RESULTS
 
Inefficient replication of wild-type HSV-1 in serum-starved normal human diploid fibroblasts at elevated temperature. Latent HSV infection is established in neurons, nondividing cells in which the major viral gene products are the latency-associated transcripts (LATs). An important function of LATs is the inhibition of apoptosis, and although the mechanism is unclear, LATs do promote the production of Hsp70 that protects cells from stress (2). LATs are expressed in a varied and cell-type-specific manner in vivo and are not produced efficiently in quiescently infected rat neuronal or human fibroblast cultures (34, 37, 50). Therefore, in an attempt to mimic this state in cultures of human fibroblasts, we serum starved NHDFs (75, 76) and preincubated these cultures at 41°C to generate nondividing human cell cultures expressing elevated levels of HSPs. These cultures could potentially accommodate higher levels of IE gene expression and therefore higher input levels of wild-type virus than previous fibroblast models (38). Cultures were preincubated at an elevated temperature for 24 to 30 h and then infected at a multiplicity of 0.5 to 1 PFU/cell. Compared to cultures at 37°C, those at 41°C expressed higher levels of Hsp70 and Hsp27 as determined by Western blotting of total cell extracts resolved by SDS-PAGE (Fig. 1A). As expected, this induction was specific to HSPs and did not affect a representative cellular antigen, PABP. Importantly infection with HSV-1 did not reduce the levels of HSP expression at either 12 h or 24 h postinfection. At 12 h postinfection, approximately 60% of cells in cultures infected at either temperature expressed the viral IE gene product ICP4, as determined by indirect immunofluorescence (Fig. 1B), confirming that temperature elevation had no effect on viral entry into the cell or the expression of at least one IE gene product. However, unlike cells infected at 37°C, where virus replicated and spread to 100% of the culture by 24 h postinfection, the percentage of cells expressing ICP4 at 41°C remained static (Fig. 1C), suggesting that the virus failed to replicate at the elevated temperature. Measuring the amount of infectious virus in cultures at either temperature confirmed that virus was actively produced in cells infected at 37°C while only minimal amounts of infectious virus were detectable in cultures at 41°C over the first 24 to 48 h and became completely undetectable by 72 h postinfection (Fig. 1D). As long as cultures were maintained at 41°C, the infection was maintained in this nonproductive state (see below).


Figure 1
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  		FIG. 1. HSV-1 infects but replicates inefficiently in NHDFs at an elevated temperature. (A) Serum-starved NHDF cells were incubated for 30 h at either 37°C or 41°C and then infected with HSV-1. Total cell extracts were prepared at 12 h and 24 h postinfection, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the indicated antiserum. The Hsp70 blot is representative of results at 12 h postinfection while Hsp27 and PABP blots are representative of results at 24 h postinfection. (B) NHDF cells grown on coverslips were serum starved and then incubated for 30 h at 41°C and infected with HSV-1 for 12 h. Fixed slides were probed with anti-ICP4 antiserum and detected with FITC-labeled anti-mouse secondary antibody. Nuclei were counterstained with Hoechst. Images were captured at a magnification of x63, and a representative field is shown. The percentage of antigen-positive cells in each field is also shown to the right. The same pattern of staining was observed at 37°C at this stage postinfection and is not shown (compare with Fig. 7). (C) Serum-starved NHDF cells on coverslips were infected at 41°C or 37°C for 24 h and processed as in panel B. The percentage of antigen-positive cells in each field is also shown to the right. (D) The amount of plaque-forming virus produced in 35-mm dishes of NHDF cells infected at 37°C (circle) or 41°C (square) at the indicated time points in days postinfection (d.p.i) was determined by titration on Vero cells and calculated as number of PFU/culture. Numbers are representative of at least three independent experiments. Inf, infected; M, mock infected.

Transient expression of a small subset of viral gene products in serum-starved NHDFs infected at an elevated temperature. Given the efficiency of infection, it was likely that population-wide changes in protein production during entry into this nonproductive state could be readily determined. We therefore examined the global pattern of proteins synthesized in uninfected and infected cells by metabolic labeling with [35S]methionine-cysteine for 1 h prior to the sampling times indicated on the figures. At 12 h postinfection cells infected at 41°C differentially expressed a small number of proteins that, based on their size and comigration with viral proteins in lytically infected cultures, appeared to represent viral polypeptides (Fig. 2A). By 24 h postinfection synthesis of these proteins had begun to decline, and by 48 h postinfection they became undetectable. Compared to cultures infected at 37°C (Fig. 2B), many of the viral proteins normally associated with productive infection were not notably synthesized in cells infected at the elevated temperature, suggesting that they were either not produced or were made at very low levels. In addition, at no point did infection at the elevated temperature alter host cell protein synthesis patterns or elicit the shutoff of host translation associated with lytic replication.


Figure 2
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  		FIG. 2. Metabolic labeling of infected NHDF cultures. (A) Serum-starved NHDFs were incubated at 41°C for 30 h and either mock infected (M) or infected with HSV-1 (Inf). At 1 h prior to the indicated sampling times in hours postinfection (h.p.i.), cultures were incubated with [35S]methionine-cysteine, and total protein was harvested by lysing cells in Laemmli buffer. Samples were resolved by SDS-PAGE, and dried gels were exposed to X-ray film. Molecular weight standards (in thousands) are shown to the left. Suspected viral proteins produced during nonproductive infection at 41°C are indicated with arrows. For ease of reference, the typical pattern of protein synthesis during productive lytic replication in NHDF cultures infected at 37°C for 24 h (from the image shown in panel B) is shown between 12 h and 24 h points. (B) Serum-starved NHDF cultures were infected as described above except at 37°C. Samples were metabolically labeled and processed as in panel A and demonstrate the expected pattern of proteins synthesized when virus replicates and spreads through the culture.

To confirm and further explore the production of viral proteins in these cells, we first performed Western blotting using antiserum against a number of viral IE proteins. Cultures at 37°C and 41°C were either mock infected or infected and then lysed in Laemmli buffer at the times indicated on the figures. Samples were resolved by SDS-PAGE, and nitrocellulose membranes were probed with antiserum, as indicated on the figures. Cells infected at either temperature expressed the 175-kDa viral IE protein ICP4, confirming results from the immunofluorescence studies shown in Fig. 1, as well as the 66-kDa IE protein ICP22 (Fig. 3A), both of which were readily visible in samples from metabolically labeled infected cell cultures at 41°C (Fig. 2). However, unlike cells infected at 37°C, where ICP22 was heavily posttranslationally modified as the infectious cycle progressed (3, 48, 49), detected as multiple immunoreactive species in samples resolved by SDS-PAGE, ICP22 expressed in cells infected at 41°C remained unprocessed, suggesting that infection was stalled at an early stage of the viral life cycle. In addition, unlike ICP4 and ICP22 the viral trans-activating protein ICP0 was produced but accumulated at significantly reduced levels compared to cultures infected at 37°C.


Figure 3
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  		FIG. 3. Viral protein production during lytic and nonproductive infection of NHDF cultures. (A) Serum-starved NHDF cultures were infected at 37°C or 41°C, and whole-cell extracts were prepared at the indicated times postinfection (h.p.i). Samples were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antiserum against the IE gene products ICP0, ICP4, and ICP22 or the late gene product Us11. Blots were also probed with antiserum toward total p38 to demonstrate even loading of samples. (B) Serum-starved NHDF cultures grown on glass coverslips were mock infected and infected at 41°C or 37°C for 24 h and then fixed and probed with anti-ICP5 antiserum. Nuclei (Nuc) were counterstained with Hoechst. Images were captured at a magnification of x20 and represent a typical field of view. Low antigen expression at 41°C resulted in a weak fluorescent signal using the same exposure times as those used for 37°C samples. Therefore, an overexposed (Overex) image of ICP5 staining at 41°C is presented in the lower panel to clearly illustrate the number of antigen-positive cells. (C) Serum-starved NHDF cultures were mock infected or infected at 41°C or 37°C for 48 h and then photographed by phase-contrast microscopy. Inf, infected; M, mock infected.

To further confirm that viral infection was not progressing in these cells, we measured the levels of Us11, a viral protein expressed late in the lytic life cycle. Compared to cells infected at 37°C and unlike abundantly expressed IE proteins, only minute quantities of Us11 were detectable in cells infected at 41°C on highly overexposed blots (Fig. 3A). This independently verified the small amounts of virus detected in cultures up to 48 h postinfection (Fig. 1D) and suggested that a small number of cells within the culture did support lytic replication to at least some level upon initial infection. To verify this and validate our observations using a different viral antigen, we examined the expression of another late viral protein, ICP5, by indirect immunofluorescence. At 24 h postinfection, while virus had spread and all cells infected at 37°C expressed extremely high levels of ICP5, an average of only 15% of cells infected at 41°C expressed this late antigen and only at low levels (Fig. 3B). Compared to cultures infected at 37°C, which exhibited significant cytopathic effect (CPE) by 48 h postinfection, those infected at 41°C remained healthy and viable, with the exception of a small number of cells that were either visibly stressed or undergoing what appeared to be cell death (Fig. 3C). While some cells may die, some of these cells may tolerate low-level virus production and release and then recover. A similar scenario has been reported in quiescently infected neuronal cell systems (71). Furthermore, death of quiescently infected cells appeared to be minimal, given the efficient recovery of virus from these cultures (see below). Overall, these observations suggested that only a portion of cells infected at the elevated temperature transiently supported virus replication and did so inefficiently while the majority of cells supported transient IE protein synthesis, which was gradually suppressed (Fig. 2) as the virus entered a nonproductive state.

At 3 and 6 days postinfection, the rates of protein synthesis (Fig. 4A) and the morphological appearance of infected cultures (Fig. 4B) were indistinguishable from those of mock-infected cells. Of interest, while expression of Us11 became undetectable even when blots were extensively overexposed, low levels of ICP4 remained even 6 days after infection, suggesting that ICP4 was either synthesized at very low levels or was proteolytically stable in cells that harbor HSV-1 (Fig. 4C). Low levels of ICP4 transcript have previously been detected in mouse ganglia latently infected with HSV (40).


Figure 4
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  		FIG. 4. Nonproductive infection is sustained at an elevated temperature and controllably recovered by exogenous expression of HSV-1 ICP0. (A) At 3 and 6 days postinfection (d.p.i.), mock-infected (M) and infected (Inf) NHDF cultures were metabolically labeled using [35S]methionine-cysteine for 1 h. Whole-cell extracts were resolved by SDS-PAGE, and then fixed and dried gels were exposed to X-ray film. The migration of molecular weight markers (in thousands) is shown to the left. (B) NHDF cultures were either mock infected (M) or infected (Inf) at 41°C, and phase-contrast images were taken after 6 days. Fluorescence in mock-infected and infected cells appeared to be serum precipitate at the elevated temperature. (C) Whole-cell extracts from NHDF cultures that were either mock infected (M) or infected (Inf) for 1 or 6 days at 41°C were resolved by SDS-PAGE and probed with antiserum against ICP4 or Us11. Images of ICP4 are taken from the same blot and exposure as presented in Fig. 3 to allow direct comparison of expression levels at days 1 and 6 postinfection. The image for Us11 is intentionally heavily overexposed to demonstrate that Us11 becomes completely undetectable over time. (D) Quiescent virus is reactivated by the expression of HSV-1 ICP0. Whole-cell extracts from NHDF cells infected for 24 or 48 h (d.p.i. 1 and 2) at 37°C, mock-infected cell extracts (M), or cell extracts from NHDF cells quiescently infected for 6 days and then reactivated (Q-R) by being infected for 48 h with an adenovirus encoding HSV-1 ICP0 (Ad0) were resolved by SDS-PAGE, and membranes were probed with antiserum against ICP0 or Us11. (E) Recovery of infectious virus from quiescently infected NHDF cultures. NHDF cells in 35-mm dishes were infected at 41°C. At 3 or 6 days postinfection (d.p.i) quiescent virus was reactivated by transducing cultures with adenovirus encoding HSV-1 ICP0, and titers of infectious virus were determined on Vero cells. Titers are representative of multiple independent experiments.

For a nonproductive infection to be considered quiescent, it must be possible to recover infectious virus from cultures in a controllable manner. To confirm that these cultures did, indeed, support quiescent rather than simply aborted infection, NHDF cultures were infected and maintained at 41°C for 6 days, 3 days to establish quiescence and a further 3 days to confirm that the state was maintained. Cultures were then returned to 37°C and transduced with an adenoviral vector encoding HSV-1 ICP0, a key regulator of reactivation from latency in vivo and quiescence in vitro (29, 30, 61, 90). At 48 h posttransduction of quiescently infected cultures, levels of the late viral protein Us11 were comparable to those observed in cells infected at 37°C at the original input dose of virus and allowed to spread for 24 to 48 h (Fig. 4d). High-level production of HSV-1 ICP0 in transduced cultures was also confirmed by Western blotting. The same immunoblot and exposure as the experiment shown in Fig. 3A were used to allow direct comparison of ICP0 expression levels during a typical lytic infection (37°C), quiescent infection (41°C), and in reactivated cultures. In addition to the restoration of Us11 production, the yields of infectious virus from ICP0-transduced cultures that had been quiescently infected for either 3 or 6 days (Fig. 4E) were equivalent to those from cells infected at 37°C (Fig. 1D) and suggested that most, if not all, of the quiescent virus harbored by these cells reactivated in response to ICP0.

When these experiments were performed with unstarved NHDF cultures, although virus replication was significantly suppressed and could be restored by exogenous provision of ICP0, a low level of virus activity was detectable in cultures even at 72 h postinfection, illustrated by small areas of CPE and detectable Us11 expression at a time when this late antigen becomes undetectable in starved cultures (Fig. 5A and B). This suggested that a mix of quiescent and replicating viruses was present, likely reflecting the mixture of dividing and nondividing cells present in unstarved cultures (76). This demonstrated that serum-starved cultures provided the optimal conditions for the establishment of quiescent infection.


Figure 5
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  		FIG. 5. Efficient quiescent infection requires nondividing cells and temperature optimization for different HSV-1 strains. (A) Unstarved NHDF cultures were either mock infected or infected with wild-type KOS for 72 h at 41°C and then photographed by phase-contrast microscopy. An image showing representative CPE evident in areas of infected cultures is shown. (B) Unstarved NHDF cultures were preincubated at 41°C and then infected with wild-type KOS for 72 h at 41°C (Inf). Total protein was then solubilized, or alternatively cultures were transduced with adenovirus encoding ICP0 (R) and then lysed 48 h later. Samples were resolved by SDS-PAGE, and membranes were probed with the indicated antiserum. (C) Serum-starved NHDF cultures were infected with wild-type HSV-1 strains KOS or Patton (Patt) at 41°C and processed as described for panel B. Note that Us11 from KOS and Patton strains exhibit differential mobility in SDS-PAGE gels. (D) Serum-starved NHDF cultures were infected with wild-type HSV-1 Patton at 42°C and processed as described in panel B. The lower panel shows an extensive overexposure demonstrating the lack of detectable Us11 protein in quiescently infected cultures. (E) The amount of infectious virus contained in NHDF cultures quiescently infected (Q) with HSV-1 Patton at 42°C at 72 h or quiescently infected cultures reactivated (Q-R) by transduction with adenovirus encoding ICP0 was determined by titration on Vero cells. Titers are representative of a number of independent experiments.

The replication of various HSV-1 strains differs markedly in sensitivity to the effects of temperature elevation. When HSV-1 Patton was tested in this system, extensive cell death and efficient production of the late viral protein Us11 were detected in cultures at 41°C, and unsurprisingly increased Us11 production was not evident in these cultures when transduced with ICP0 (Fig. 5C). However, by simply raising the temperature to 42°C, all of the characteristics of quiescent infection described for HSV-1 KOS were restored using the Patton strain, as illustrated by the pattern of Us11 expression (Fig. 5D) and levels of infectious virus present (Fig. 5E) in quiescent and reactivated cultures. As this system relies solely on temperature to suppress virus replication, these results highlight the importance of optimizing temperatures for individual HSV-1 strains.

Specific reactivation of quiescent virus by ICP0. We then examined ICP0-mediated reactivation more closely in this system. Quiescence was established by infecting cells at 41°C (KOS strain) for 72 h, and then uninfected or quiescently infected cultures were returned to 37°C and either mock transduced or transduced with adenoviral vectors encoding the HSV-1 IE gene products ICP4 or ICP0. ICP4 is a key transcription factor required for progression of the viral life cycle during lytic replication but has been reported not to induce virus reactivation, while ICP0 is a multifunctional gene trans-activator and important regulator of HSV reactivation (29, 30, 61, 90). At 48 h posttransduction cultures were examined for CPE or metabolically labeled with [35S]methionine-cysteine to examine the pattern of protein synthesis in cultures. Figure 6A shows that lytic patterns of viral protein synthesis were restored in quiescently infected cultures transduced with HSV-1 ICP0 but not in mock- or HSV-1 ICP4-transduced cultures. Cultures that were not previously quiescently infected with HSV-1 were also transduced with the same adenoviral vector expressing HSV-1 ICP0 and demonstrated that the reappearance of HSV-1 in cultures did not originate from any source other than the initial experimental quiescent infection. While NHDF cultures that were not initially quiescently infected with HSV-1 exhibited no morphological changes in response to adenovirus encoding ICP0, quiescently infected cultures exhibited extensive CPE associated with lytic replication (Fig. 6B). In quiescently infected cultures that were transduced with an adenoviral vector encoding ICP4, the vast majority of cells remained morphologically indistinguishable from uninfected NHDFs while a small percentage of cells appeared rounded 48 h after return to 37°C. Small amounts of infectious virus produced in these cultures (at levels as high as 103 PFU/culture) along with the inability to detect significant amounts of viral protein synthesis by [35S]methionine-cysteine labeling suggested that these represented cells that had spontaneously reactivated.


Figure 6
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  		FIG. 6. HSV-1 ICP0 specifically reactivates virus from quiescently infected NHDF cells. Serum-starved NHDF cultures were mock infected (M) or infected (Inf) at 41°C for 72 h to establish quiescence. Cultures were returned to 37°C and either mock transduced with growth medium (med) or adenoviral vectors encoding HSV-1 ICP4 (Ad4) or HSV-1 ICP0 (Ad0) as indicated. (A) At 47 h postinfection cultures were metabolically labeled for 1 h using [35S]methionine-cysteine, whole-cell extracts were resolved by SDS-PAGE and fixed, and dried gels were exposed to X-ray film. The migration of molecular weight markers (in thousands) is indicated to the left. (B) Phase-contrast images of mock-infected or quiescently infected NHDF cultures 48 h posttransduction with the indicated adenoviral vectors described in panel A.

Approximately 60% of cells were initially infected, as determined by immunofluorescent staining for viral ICP4 (Fig. 1), yet results shown in Fig. 6 suggested that all cells in ICP0-reactivated cultures were supporting lytic replication. It was likely, therefore, that part of this effect was due to secondary spread of reactivated virus, similar to virus spread in cultures infected at 37°C, as shown in Fig. 1. To determine the true degree of reactivation, we performed the same experiment in the presence of HS to neutralize secondary spread of virus in cultures, as used in previous studies (61). We first determined that medium containing 5% HS eliminated infectivity of HSV-1 in the range of infectious virus produced by our quiescent systems (data not shown). Quiescently infected NHDFs were reactivated by transduction of cultures with the adenoviral vectors, as indicated on the figures, for 2 h; adenovirus was then removed, washed, and replaced with either growth medium containing 5% FBS or 5% HS. At 47 h postinfection cultures were metabolically labeled for 1 h with [35S]methionine-cysteine. Total cell lysates were resolved by SDS-PAGE, and fixed dried gels were exposed to X-ray film. The presence of HS resulted in a mixture of host cell and robust viral protein synthesis (Fig. 7A), suggesting that HS prevented the secondary spread of virus in reactivated cultures and that a large proportion of the culture initially infected reactivated in response to ICP0. The effectiveness of HS in neutralizing HSV-1 was directly confirmed at the end of this assay by the fact that infectious virus was undetectable in the supernatants of quiescent cultures reactivated in the presence of HS, while those reactivated in the presence of FBS produced titers in the expected range of approximately 106 to 107 PFU/culture (not shown). In agreement with these observations Western blotting of samples with anti-Us11 antiserum showed a partial decrease in production of this protein in cultures reactivated in the presence of HS (Fig. 7B). As mentioned previously, low levels of spontaneous reactivation occurred in control cultures, and this was further evident in the low-level production of Us11 in mock- or ICP4-transduced quiescently infected cultures 2 days after the return to 37°C. However, this was largely due to secondary virus spread from a small number of cells in which spontaneous reactivation had occurred as HS dramatically reduced the amount of Us11 found in quiescently infected cultures to practically undetectable levels (Fig. 7B). To further assess this, we examined the effects of HS using indirect immunofluorescence to determine the pattern of expression of the late gene product, ICP5 (Fig. 7C). While 95 to 100% of quiescently infected cells expressed ICP5 to various levels of intensity when reactivated with ICP0, as expected this percentage dropped to about 60% when reactivated in the presence of HS. Quiescently infected cultures that were not reactivated with ICP0 and were maintained in the presence of HS showed that about 2% of cells underwent spontaneous reactivation in the first 48 h after the return to 37°C.


Figure 7
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  		FIG. 7. HS prevents secondary virus spread and illustrates the efficiency of virus reactivation from quiescently infected NHDFs. (A) Serum-starved NHDFs were infected at 41°C for 72 h and then returned to 37°C and transduced with the indicated adenoviral vectors encoding ICP4 (Ad4) or ICP0 (Ad0). An additional Ad0-transduced culture was maintained in the presence (+) of 5% HS after the adenoviral vector was removed to prevent secondary spread of reactivated virus. At 47 h postinfection cultures were metabolically labeled; whole-cell lysates were resolved by SDS-PAGE and fixed, and dried gels were exposed to X-ray film. Migration of molecular weight standards (in thousands) is indicated to the left. (B) NHDF cells were either mock infected (M) or infected (Inf) at 41°C for 72 h and then returned to 37°C and mock transduced with growth medium (Med) or transduced with the indicated adenoviral vectors in the presence or absence of HS as described in panel A. At 48 h posttransduction whole-cell extracts were resolved by SDS-PAGE, and membranes were probed with antiserum against Us11. Two different exposures of the same blot are provided to allow quantitative comparison between samples. Exp, exposure. (C) NHDF cells grown on glass coverslips were infected at 41°C and mock transduced (Med) or transduced with adenovirus encoding ICP0 (Ad0) in the presence or absence of HS. At 48 h posttransduction cultures were fixed and probed with the antiserum against ICP5. Nuclei were counterstained with Hoechst, and images were captured at a magnification of x20. Two representative fields are shown for HS experiments, demonstrating the natural variation in the distribution of quiescently infected cells in Ad0-reactivated cultures and the very low incidence of spontaneous reactivation in cultures mock transduced with growth medium (Med) in the presence of HS. Percent reactivation is presented to the right of the immunofluorescent images.

Enlargement of PML structures in the nuclei of quiescently infected NHDFs. The experiment shown in Fig. 3A demonstrated that the viral trans-activator ICP0 accumulated at significantly reduced levels in cells infected at 41°C. An extensive body of work from a number of laboratories has shown that ICP0 plays an important role in initiating productive infection by dispersing repressive nuclear ND10 structures containing PML (5-7, 16, 18-20, 22, 23, 35, 44, 45, 47, 62, 70). We therefore examined the localization pattern of PML during the establishment of quiescence as both a readout for ICP0 function and a potential mechanism for repression of virus replication. Indirect immunofluorescence showed that PML structures exhibited a normal pattern of nuclear speckling in uninfected cells at 41°C, and dispersal of these structures was readily observed in infected cells undergoing lytic replication at 37°C, as identified by costaining for the viral antigen ICP4 (Fig. 8). This showed that PML structures are disrupted during lytic infection of serum-starved NHDF cultures, as reported in other cell types infected with HSV-1. However, cells infected at 41°C contained dramatically enlarged PML structures, and this occurred only in infected cell nuclei that costained for viral antigen while neighboring uninfected cells retained normal staining patterns. These findings support previous suggestions using IE mutant viruses that PML rearrangement is a direct cellular response to incoming virus that must be overcome to establish a productive infection.


Figure 8
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  		FIG. 8. Enlargement of PML structures in NHDF cells infected at elevated temperature. Serum-starved NHDF cultures grown on glass coverslips were mock infected and infected at 41°C or 37°C for 10 h and then fixed and coprobed with antiserum against human PML (green) and HSV-1 ICP4 (red); proteins were then detected using the appropriate FITC-conjugated or tetramethyl rhodamine isothiocyanate-conjugated secondary antibodies. Nuclei were counterstained with Hoechst.


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DISCUSSION
 
Latent infection by HSV-1 is a complex state that is relatively poorly understood, due in part to the difficulties in modeling the process in vitro to facilitate studies at the cellular level. The difficulties in establishing efficient quiescent infection of human cells have been overcome by disrupting ICP0 directly, indirectly, or in combination with other viral IE gene products (18, 21, 30, 31, 36, 57, 58, 61, 63, 69). Whether quiescence established by wild-type virus follows a similar process to IE mutants and what mechanisms of host restriction might accommodate this process have remained unclear. Here, we have described simple culture conditions that supported the establishment of a nonproductive infection in human cells using wild-type HSV-1 that displayed key properties of the latent or quiescent state defined in previous models, providing a convenient means of studying quiescent infection using unmodified virus.

Entry into quiescence in this system was characterized by the failure to shut off host translation, together with robust synthesis of some IE proteins and low-level viral replication, both of which were rapidly suppressed as the virus entered the nonproductive state. Expression of viral gene products has been reported during latency in vivo and entry into quiescence in neuronal models (1, 17, 18, 40, 66, 74). In addition, the pattern of proteins actively synthesized during entry into quiescence in our model was strikingly similar to that observed in neuronal cell lines that are nonpermissive for HSV-1 replication (51), while low-level lytic replication preceding entry into the nonproductive state has been reported in neuronal cell systems (71), likely reflecting a natural part of the dynamic process of establishing latent infection in vivo.

Although some IE proteins are produced upon infection at the elevated temperature, the failure to process ICP22 and inefficient production of ICP0 are likely to dramatically hinder the ability of the virus to establish a productive infection. IE gene expression and the formation of viral replication compartments initiate at nuclear structures within the host cell known as ND10 or PML bodies that are disrupted by ICP0 to establish a productive infection (5, 7, 16, 19, 20, 23, 35, 44, 45, 47). In neuronal lines that are less permissive for HSV-1, these structures are enlarged, and ICP0-defective viruses become quiescent (33) while HSV-1 mutants defective in the synthesis of a number of IE gene products including ICP0 establish quiescent infection of human fibroblast lines, resulting in the accumulation of individual, enlarged PML bodies (21). The finding that IE mutants can establish quiescent infection in vitro and latent infection in vivo has led to the suggestion that entry into a nonproductive state is a passive process that requires little or no viral function. This is very likely to be the case, but cell types that accommodate quiescent infection with wild-type virus must therefore suppress the expression or function of these IE proteins that normally regulate commitment to lytic replication. Indeed, cells lacking HCF and Oct-1, critical components of VP16 complexes that initiate IE gene expression, are poorly infected by HSV-1 (54, 88). The significantly reduced accumulation of ICP0 during entry into quiescence, likely below a required threshold level to disperse PML structures, suggests that wild-type virus enters quiescence in our model in the same or a very similar manner to that reported for IE mutants, but expression of other IE gene products suggests that this process does not occur through general suppression of VP16 function. Determining how these cells suppress ICP0 without altering production of other IE proteins may provide important insights into the mechanics of how certain host cell types repress lytic replication of wild-type virus resulting in quiescent infection. Of additional interest, a report that the cytotoxicity of certain mutants is reduced by the absence of ICP0 (64) suggests that the ability of serum-starved NHDFs to accommodate higher inputs of wild-type virus might also relate to their ability to suppress the accumulation of ICP0.

Quiescent virus was readily recovered in this system upon transduction of cultures with ICP0, a critical regulator of lytic replication and reactivation. Although some reports have shown that virus can be reactivated under certain conditions in the absence of ICP0 (46, 56), IE mutants that form quiescent infection are invariably defective in ICP0 expression, directly or indirectly, and it is generally accepted that ICP0's activity is a key regulator of virus reactivation. Notably, exogenous expression of ICP4 did not result in reactivation of quiescent virus above spontaneous levels in our system. This was not surprising, given that ICP4 was efficiently expressed during entry into quiescence and has been reported previously not to reactivate quiescent HSV in human cell line models (29, 30, 90). ICP0, ICP4, and VP16 have been reported to reactivate virus in a neuronal model, with ICP0 reactivating virus more efficiently (26, 46). In addition, a range of stress-inducing agents also reactivates virus in certain neuronal models while quiescence in fibroblasts is frequently refractory to most reactivation stimuli except ICP0. This has led to the suggestion that the viral genome is maintained in a more repressed state in human fibroblasts than in nonhuman neuronal cell lines, and to date our findings are in line with these previous reports. When cultures were returned to 37°C, removing the selective pressure to maintain the quiescent state, low levels of spontaneous reactivation occurred. This phenomenon is widespread in quiescent systems and likely reflects the natural dynamic state of the in vivo situation. Spontaneous reactivation also occurs in mouse models of HSV latency but is contained by local immune activity (24, 42), and virus is frequently shed from asymptomatic individuals infected with HSV-1 (25), suggesting that low levels of viral activity in latently infected cultures simply reflect a natural phenomenon that cannot be contained in the absence of an immune system. Along these lines, the addition of human serum to quiescently infected cultures upon downshift to 37°C neutralized the spread of virus from this small population undergoing spontaneous reactivation. All quiescently infected cells may gradually reactivate in an uncontrollable manner, but we are currently testing the possibility that quiescent cultures may be maintained for more extended periods at 37°C by culturing in the presence of HS.

Our preliminary data suggest that changes in the levels and phosphorylation of cellular proteins are readily detectable on a population-wide scale in this system, similar to a large number of biological systems used at similar efficiencies. In addition, the use of HS to prevent virus spread should also allow us to distinguish and study secondary events after virus reactivation. To date, we have found that quiescent infection is accommodated by these cultures to the point where practically all cells are infected but at higher multiplicities; although quiescent infection is still established with the same kinetics and with a very similar phenotype, cells begin to exhibit signs of stress (unpublished observation). A similar situation has been reported in neuronal cell systems infected at a multiplicity of 1 PFU/cell (4), suggesting similarities in the limits to input virus prior to the onset of cellular stress. This may be due to excess production of IE proteins, known to be toxic when overproduced (38), or to additional stresses caused by increasing amounts of noninfectious particles that comprise the ratio of particles to PFU of standard laboratory virus stocks. Regardless, given the simplicity and efficiency of this system, it should provide a relatively easy to use complement to existing models and allow detailed biochemical analysis of quiescent infection of human cells by wild-type HSV-1.


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ACKNOWLEDGMENTS
 
We thank Ian Mohr, Saul Silverstein, Richard Roller, and John Blaho for generously providing reagents, together with Martin Clynes, Finbarr O'Sullivan, and members of the National Institute for Cellular Biology for their support.

This work was supported by a grant from the Science Foundation Ireland (06 IN.1 B80) to D.W.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. Phone: 353 1 7006260. Fax: 353 1 7005484. E-mail: derek.walsh{at}dcu.ie Back

{triangledown} Published ahead of print on 13 August 2008. Back


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Journal of Virology, October 2008, p. 10218-10230, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00859-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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