The Role of Cyclic Nucleotide Mediators in Latency ...

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Boston, Massachusetts, USA. Summary ... Infirmary, 243 Charles St, Boston, MA 0 2 1 14 , USA. ... NJ). isoproterenol HCl (Winthrop-Beron. Lab, New York.
Eye (1991) 5, 627-635

The Role of Cyclic Nucleotide Mediators in Latency and Reactivation of HSV-l Infected Neuroblastoma Cells ALEJANDRO RODRIGUEZ, MAITE SAINZ DE LA MAZA, JOHN MISSRY, C. STEPHEN FOSTER Boston, Massachusetts, USA

Summary The mechanisms that control herpes simplex virus type

1 latency and reactivation

are still poorly understood. We developed an in vitro murine neuroblastoma cell HSV-infected, acyclovir suppressed model to study the influence of different cyclic nucleotide mediators on the latency and reactivation of HSV-l. A positive cDNA 'in

situ' hybridisation for HSV genome was used to prove the establishment of a viral­ host cell nuclear relationship. An ABC-immunoperoxidase reaction to cell surface HSV mature glycoproteins was also performed to determine the time of viral reactiv­ ation with formation of mature virions. Supernates of cultured cells were placed on Vero cells for confirmation of reactivation by classic cytopathic effect. Theophylline

(SO [!g/ml) and dibutyril-cAMP (0.1, 0.5, 1 mg/ml) produced the most pronounced 150%. Epinephrine (10, 20 [!g/ml) had an intermediate effect on accelerating viral reactivation; and verapamil (20, SO [!g/ml), theophylline and epinephrine at lower doses had a smaller effect. Car­ bamylcholine (10 [!g/ml) prolonged the time to viral reactivation by 100%, 36 hours compared to control time of 18 hours. Insulin (0.1,0.5,1 mg/ml) also prolonged HSV

response, accelerating HSV reactivation time by

'latency' by six hours. Exogenous dibutyril-cGMP and carbamylcholine at lower concentrations did not have an effect on viral reactivation. These findings suggest that there is a relationship between changes of intracellular concentrations of cyclic nucleotides and HSV latency and reactivation.

Latently infected trigeminal ganglia have been thought to be the source of virus for recurrent herpetic keratitis in humans, a major cause of corneal blindness in developed countries. 1 Current antiviral therapy has proved ineffective in preventing the estab­ lishment of latency. The molecular details of the relationship between virus and host cell DNA during the latent state have not been

definitely elucidated. 2•3 An alternative thera­ peutic strategy to viral DNA elimination from latently infected ganglia might include efforts to indentify and manipulate factors involved in the control of mechanisms responsible for the induction of and reactivation from the latent state. A variety of physiological (fever, trauma, sunburn, menses, stress) and experi­ mental (epinephrine, prostaglandins, ultra-

From: Hilles Immunology Laboratory, Massachuesetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA. Supported by the Susan Morse Hilles Fund. Correspondence to: C. Stephen Foster, MD. , Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 0 2 1 14 , USA.

628

ALEJANDRO RODRIGUEZ ET AL

violet light) stimuli can be associated with HSV-l reactivation in patients and in experi­ mental models respectively.4- 1 II These 'trig­

gers' may influence, through the action of first order messengers such as catecholamines

Park, NJ) were used to grow and maintain the cell lines in an incubator at 37°C and 5'X, CO, during the experiments.

Mediators: Mediators that increase intracel­

and/'Jf arachidonic acid metabolites, cyclic adenosine monophosphate (cAMP) or cyclic

lular levels of cAMP: N6-2'-O-dibutyrilade­

trations within latently infected neurons. We

epinephrine (Elkins-Sinn. Inc.. Cherry Hill,

nosine 3',5' cyclic'monophosphate sodium

guanosine monophosphate (cGMP) concen­

salt

have already shown in an in vivo and in vitro murine trigeminal ganglion latency model

NJ).

cAMP and cGMP may influence maintenance

maceuticals. Whippany. NJ). Mediators that

(purity

=

97%) (Sigma, St Louis, MO),

isoproterenol

HCl

(Winthrop-Beron

Lab, New York. NY). theophylline (Elkins­

that the relative proportions of intracellular

Sinn. Inc.) and verapamil HCI (Knoll Phar­

of (elevated cGMP with no change in cAMP

increase intracellular levels of cGMP: N2,2'­

latency.II

phate sodium salt (purity-96°/c)

levels) or reactivation from (elevated cAMP) In

order

to

study

further

the

O-dibutyrilguanosine 3"5' cyclic monophos­ (Sigma), car­

relationship between intracellular neuronal

bamylcholine (Alcon Inc., Humacao. Puerto

studied the effects of putative mediators of

(Eli Lilly and Co .. Indianapolis. IN). Other

cyclic nucleotide levels and HSV latency we

Rico) and regular recombinant-DNA insulin

intracellular cyclic nucleotide levels on an

mediators used: sodium cromoglycate (Fisons

infected, acyclovir-suppressed model using

sodium phosphate (Elkins-Sinn. Inc.), cyclos­

detection) and avidin-biotin immunoperoxi­

Hanover,

in vitro murine neuroblastoma cell HSV­

cDNA 'in situ'

hybridisation (viral DNA

dase (mature virion detection) techniques to determine the time of viral reactivation (posi­

tive cDNA 'in situ' hybridisation, positive

ABC

immunoperoxidase)

from

'latency'

(positive cDNA in situ' hybridisation, nega­ '

tive ABC immunoperoxidase) for each medi­

ator. These findings may have implications for clinical ocular HSV recurrent infections.

Corp.,

Bedford.

MA).

dexamethasone

porin A (Sandoz. Pharmaceutical Corp., East 2 NJ). 1

Experiment design: Neuroblastoma cells were trypsinised and plated into 6-well, 9.62 cm2

flat bottom tissue culture plates (Becton­

Dickinson 10.5

x

contammg

Labware)

a

22 mm Thermanox tissue culture plas­

tic coverslip (Miles Lab Inc .. Naperville, II). Each experiment was performed with its own

set of controls and was replicated at least four

Material and Methods

times to demonstrate the reproducibility of

Cell lines: C-1300 murine neuroblastoma cells

the results.

ville, MD) were cultured using Nutrient Mix­

HSV-J infection of neuroblastoma cells: After

(American tissue Culture Collection, Rock­

ture F-12 with L-glutamine media (Gibco Laboratories,

Grand

Island,

NY)

sup­

plemented with 5% qualified fetal bovine

three days of cell incubation of 37°C, 5% CO2, the cultures were infected with 1 mllwell of HSV-l KOS strain (titer

=

2

x

10K PFU/ml,

serum (FBS) heated and inactivated at 60°C

from Dr David Knipe.

5,000 mcglml of streptomycin (Gibco Labs),

ent Mixture FO-12 to an MOl of 2. for one

Inc., McLean VA), and 250 mg of gentamicin

and the wells were washed and rinsed three

for

one

hour,

5,000 U/ml

of

penicillin,

250 mcglml of fungizone (Squibb Flow Labs,

sulfate (Gibco Labs). Vero cells from monkey

kidney (CCL8!, ATCC) were cultured with

Minimum Essential Medium (MEM) with

Earle's salts (Gibco Labs), 5% FBS and peni­ cillin-streptomycin-fungizone-gentamicin sol­ ution

as above. 75

cm' sterile cell culture

flasks (Becton-Dickinson Labware, Lincoln

Harvard Medical

School, Boston. MA) diluted with FBS Nutri­

hour. The viral suspension was then removed

times with FBS Nutrient Mixture F-12 fol­

lowed by a post-infection incubation period of six hours with 5% FBS Nutrient Mixture F-12.

Slippression of HSV-J with acyclovir: After

the

post-infection

HSV-l

incubation

period, the

infected neuroblastoma cells were

THE ROLE OF CYCLIC 'JCCLEOTlDE MEDIATORS IN LATENCY AND REACTIVATION OF HSV-J LATENCY

treated with 10 [lg/ml of acyclovir (ACV ) (Burroughs-Welcome,

Res. Triangle Park,

NC) and 1 [lg/ml of 7S-Nerve Growth Factor (7S-NGF)

(Collaborative

Research

629

applied for 10 minutes after removal of the

glass coverslip. The coverslips were rinsed with wash buffer for 10 seconds and the detec­

Inc.,

tion reagent (avidinbiotinylated horseradish

induce suppression of HSV-l . U At the end of

coverslips were rinsed with wash buffer, coun­

Bedford, MA) everyday for four days to day four, the cells were washed and rinsed three times with FBS Nutrient Mixture F-12 to remove the ACVnS-NGF solution.

ation time: Cyclic nucleotide mediators at dif­

doses

HSV-infected,

were

then

added

to

terstained with fast green FCF and mounted with gelvatol for light microscopy.

ABC immllnoperaxidase staining: The other

Time-course for detection of HSV-J reactiv­

ferent

peroxidase) was applied for 10 minutes. The

the

half of the coverslip was also rinsed in PBS (pH 7.2) and fixed with acetone as above.

Coverslips were incubated with a 5% normal

ACV-suppressed neuroblas­

goat serum solutions for 30 minutes. A rabbit

(unreported) were performed to determine

Corp., Santa Barbara, CA) was diluted to

consisting of the removal of the supernate and.

microfilter

tivation on Vero cellsl� and immunohisto­

cells for 15 minutes on a rotating table. A

time course was 4i1 to 72 hours depending on

centrifugation with 2800 rpm for 10 minutes at

Controls: These consisted of neuroblastoma

room temperature in a moisture chamber.

without

with PBS (pH 7.2) for 10 minutes. HP2

toma cells. Preliminary dose-response studi.es the mediator toxicity limits. A time-course a set of coverslips every two hours for cocul­

chemical staining respectively was done. The the type of mediator used.

1: 100 with

1%

BSA, filtered with a 0.2 [lm

(Gelman

Sciences

Inc.,

Ann

Arbor, MI) and absorbed with neuroblastoma

further dilution to 1:1600 with 1% BSA and

25°C was followed by adding it to the cover­

slips for 45 minutes (primary incubation) at

cells on coverslip without HSV infection and ACVnS-NGF

anti-HSV-l antibody (IgG anti-HSV , Dako

(positive-control);

and a set of coverslips HSV-infected, ACV­

The coverslips were then rinsed three times

0.3'};" was added to each coverslip for 30 min­

culture

utes. A rabbit ABC Vectastain kit (Vector

alone, which were withdrawn along with the

used to follow the staining. The secondary

(model control).

coverslip; one drop of goat anti-rabbit [gG

suppressed,

incubated

with

fresh

media and nerve growth factor (1 �lg/ml) mediator coverslips during every time-course

eDNA

'in

situ'

Laboratories [nc., Burlingame, CA) was then

incubation was performed after rinsing the

biotinylated antibody was applied for 45 min­ hybridisation:

Withdrawn

coverslips were left to dry at room temper­

utes, followed by a rinse with PBS. The ter­

tiary

45

minute

incubation

was

the

avidin-biotinylated

Diagnostics, Inc., New York, NY ). Therma­

aminoethycarbazole substrate for 20 minutes.

with an HSV cDNA probe assay (ENZO

peroxidase

with

ature for at least two days and then stained

per­

formed before reacting the coverslips with the

nox coverslips were cut into two equal pieces;

Coverslips were rinsed with acetate buffer

stain; the other was rinsed with PBS (pH 7 .2)

glacial acetic acid was applied before coun­

one was used for the ABC immunoperoxidase and fixed with 100% acetone for 10 minutes.

One drop of the DNA probe reagent was then

applied to each Thermanox tissue culture

coverslip and a glass coverslip was then placed

(pH 5.0) and fixed with 4% formaline; 1%

terstaining

with

Gill's

hematoxylin

and

mounting with gelvatol for light microscopy.

Cocultivation of supernates on Vera cells: All

to cover the preparation which was then

supernates from the time-course (mediator

utes. After removing the coverslip from the

and placed on confluent monolayers of Vero

placed onto a 92°C heating block for 3-4 min­

block, hybridisation proceeded at room tem­

perature for 20 minutes in a moisture cham­ ber.

The

post -hybridisation

reagent

was

samples and controls) coverslips were filtered

cells and observed daily for 10 days, using an

inverted light microscope for cytopathic effect (CPE).

630

Fig. 1.

ALEJANDRO RODRIGUEZ ET AL.

Positive eDNA 'in situ'

hybridisation for HSV in HSV- 1 infected neuroblastoma cells (4 0x)

Statistics: Differences in time for HSV-l reac­ tivation (positive ABC immunoperoxidase staining) for each mediator compared to con­ trol were analysed using the Analysis of Vari­ ance test (p