Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation

Heba H. Mostafa,

*

Thornton W. Thompson,

*

Adam J. Konen,

Steve D. Haenchen,

*

Joshua G. Hilliard,

Stuart J. Macdonald,

Lynda A. Morrison,

David J. Davido

a_{Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA}

b_{Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis,}

Missouri, USA

ABSTRACT

In the process of generating herpes simplex virus 1 (HSV-1) mutations

in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a

vi-ral mutant, termed KOS-NA, that was severely impaired for acute replication in the

eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection,

and reactivated poorly from explanted TG. To identify the secondary mutation(s)

re-sponsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA

genome and noted that it contained two nonsynonymous mutations in

UL39

, which

encodes the large subunit of ribonucleotide reductase, ICP6. These mutations

re-sulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950)

substi-tutions in ICP6. To determine whether alteration of these amino acids was

re-sponsible for the KOS-NA phenotypes

in vivo

, we recombined the wild-type UL39

gene into the KOS-NA genome and rescued its acute replication phenotypes in

mice. To further establish the role of

UL39

in KOS-NA’s decreased pathogenicity,

the

UL39

mutations were recombined into HSV-1 (generating UL39

), and this

mutant virus showed reduced ocular and TG replication in mice comparable to that of

KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative

to the wild-type protein. Moreover, we observed that KOS-NA does not counteract

caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies

with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely

plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently

impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.

IMPORTANCE

HSV-1 is a major human pathogen that infects

⬃

80% of the human

population and can be life threatening to infected neonates or

immunocompro-mised individuals. Effective therapies for treatment of recurrent HSV-1 infections are

limited, which emphasizes a critical need to understand in greater detail the events

that modulate HSV-1 replication and pathogenesis. In the current study, we

identi-fied a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in

the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also

known as ICP6). This mutant form of ICP6 was responsible for the attenuation of

KOS-NA

in vivo

and resulted in diminished ICP6 protein levels and antiapoptotic

ef-fect. Thus, we have determined that subtle alteration of the UL39 gene regulates

ex-pression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially

making KOS-NA a promising vaccine candidate against HSV-1.

KEYWORDS

HSV-1, ICP6, UL39, antiapoptosis, pathogenesis, ribonucleotide

reductase, viral mutant

Received18 October 2017Accepted3 January 2018

Accepted manuscript posted online10 January 2018

CitationMostafa HH, Thompson TW, Konen AJ, Haenchen SD, Hilliard JG, Macdonald SJ, Morrison LA, Davido DJ. 2018. Herpes simplex virus 1 mutant with point mutations inUL39is impaired for acute viral replication in mice, establishment of latency, and explant-induced reactivation. J Virol 92:e01654-17.https://doi .org/10.1128/JVI.01654-17.

EditorRichard M. Longnecker, Northwestern University

Copyright© 2018 American Society for Microbiology.All Rights Reserved. Address correspondence to David J. Davido, ddavido@ku.edu.

*Present address: Heba H. Mostafa, Clinical Microbiology Laboratories, University of Rochester Medical Center, Rochester, New York, USA; Thornton W. Thompson, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA; Steve D. Haenchen, University of Arizona College of Public Health, Tucson, Arizona, USA.

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H

erpes simplex virus 1 (HSV-1) is the primary cause of infectious blindness in

western industrialized countries. The virus alternates between two phases during

its life cycle: lytic and latent (1). The lytic replication cycle, which occurs in epithelial

cells of the cornea or the mucocutaneous regions of the oral labial region, is

charac-terized by a temporal cascade of immediate early (IE), early (E), and late (L) gene

expression (2). Latency, on the other hand, is established in sensory neurons that

innervate the primary site of lytic infection. During latency, neither productive infection

nor significant viral gene expression occurs, with the exception of the

latency-associated transcripts (LATs) (3). Reactivation of the latent viral genome can be

trig-gered under various conditions of stress. IE genes are the first to be expressed, and

among these is the gene for the viral protein infected cell protein 0 (ICP0).

ICP0 is a multifunctional protein that has important functions in acute replication,

establishment of latency, and reactivation (4, 5). We examined how phosphorylation of

HSV-1 ICP0 in 3 specific regions of the protein affects its functions, and viral replication,

by creating the viral mutants Phos 1, 2, and 3 (6). When these mutants were

charac-terized using the mouse ocular model of HSV-1 latency and reactivation, Phos 1 and 2

had attenuated phenotypes

in vivo

, whereas Phos 3 behaved like wild-type (WT) HSV-1

in these assays (7). During the course of generating the Phos 3 mutant, we isolated a

Phos 3 variant (termed KOS-NA) that, in contrast, was severely impaired in its acute

replication in mouse eyes and trigeminal ganglia (TG), significantly reduced in its

capacity to establish latency, and reactivated poorly when latently infected TG were

explanted in culture. Given the severe attenuation of KOS-NA relative to Phos 3 in mice,

we hypothesized that KOS-NA contained a secondary mutation(s) in one or more key

viral genes that regulate HSV-1 pathogenesis. Furthermore, such secondary mutations

would provide insight into HSV-1 pathogenesis and could be useful in developing

therapeutic interventions against HSV-1 (e.g., vaccine).

To understand the genetic basis for KOS-NA’s

in vivo

phenotypes, we sequenced its

genome and compared it to that of its wild-type parental virus, strain KOS (8, 9). We

discovered, besides ICP0’s phosphorylation mutations, two nonsynonymous mutations

in the ICP4 gene, one nonsynonymous mutation in the glycoprotein I (gI) gene, and two

nonsynonymous mutations (L393P and R950H) in the UL39 gene of the KOS-NA

genome. Given the parallels between the

in vivo

replication phenotypes of published

UL39

mutants and KOS-NA (10–12), we proposed that at least one of the mutated

codons in

UL39

of KOS-NA is responsible for its attenuated phenotypes.

UL39

encodes

the large subunit of ribonucleotide reductase, also known as ICP6. The ribonucleotide

reductase enzyme complex is essential for viral DNA replication because it converts

ribonucleotides to deoxyribonucleotides (13, 14). Other key functions of ICP6 have

been associated with inhibiting apoptosis (15) and activating or inhibiting necroptosis

(16, 17). Although this gene is not essential for viral growth and replication in dividing

cell lines, its function is required for viral replication in quiescent cells, such as neurons

(18, 19), and several studies have shown that

UL39

mutants are severely impaired for

replication, establishment of latency, and/or reactivation in murine models of HSV-1

infection (10, 11). Based on these observations, we wanted to test whether KOS-NA’s

attenuation is related to its

UL39

mutations.

To investigate this possibility, we rescued the mutated UL39 gene within the

KOS-NA genome with a wild-type copy of

UL39

(KOS-NAR), and we independently

introduced the mutated copy of the

UL39

from the KOS-NA genome into the wild-type

KOS genome (UL39

). The acute replication of both viruses in the eyes and TG of

mice, as well as establishment and reactivation from latency, was examined.

RESULTS

Mutations in KOS-NA impair acute viral replication in the eyes and TG.

We

initially examined acute replication of KOS-NA at the periphery in ocular epithelia. For

these experiments, CD-1 mice were infected with 2

⫻

10

PFU of wild-type HSV-1 (KOS),

the attenuated ICP0 null mutant 7134, or KOS-NA per eye. 7134 was used as an

ICP0-null, attenuated control virus in these experiments (20). Eye swabs from mice were

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taken at days 1, 3, 5, 7, and 9 postinfection. As shown in Fig. 1A, the levels of replication

of KOS-NA on days 1 and 3 postinfection were 79-fold (

t

test,

P

⫽

0.015) and 25-fold (

t

test,

P

⫽

0.006) lower than that of KOS, respectively. On day 5 postinfection, no KOS-NA

infectious virus was detected, whereas KOS still replicated to levels

ⱖ

1,000-fold above

the limit of detection (

t

test,

P

⫽

3.9

⫻

10

) and continued to replicate through 7 days

postinfection. Interestingly, the replication of KOS-NA was more impaired than that of

7134 on days 3, 5, and 7 postinfection. When acute replication in neurons of the

trigeminal ganglia (TG) was examined, KOS-NA showed no detectable replication at all

time points examined. Relative to KOS titers, these represented decreases of up to

5,000-fold on days 3, 5, and 7 postinfection (Student’s

t

test,

P

⫽

1.4

⫻

10

to 3.5

⫻

10

). 7134, as expected (7), replicated in TG neurons of CD-1 mice, albeit poorly

(Fig. 1B).

Mutations in KOS-NA reduced the establishment of latency.

To quantify the

relative amount of viral DNA present in latently infected neurons, TG were collected at

days 28 to 30 postinfection and assayed for the presence of HSV-1 DNA using

quantitative real-time PCR. As shown in Fig. 2, the amounts of latent viral DNA present

in TG were significantly reduced for KOS-NA (53-fold; one-way analysis of variance

[ANOVA],

P

⬍

0.05) and 7134 (12-fold; one-way ANOVA,

P

⬍

0.05) relative to the level

for KOS. Decreases in the establishment of latency for KOS-NA are probably linked to

its impaired replication during acute infection in the TG.

FIG 1Acute replication of KOS-NA in mice. Groups of mice were infected with 2⫻105_{PFU per eye. Tear}

film was collected on days 1, 3, 5, 7, and 9 postinfection, and TG were collected at the same time points from cohorts. Infectious virus in swab samples and homogenized TG was quantified by plaque assay. (A) Acute corneal replication of KOS-NA. (B) Acute replication of KOS-NA in TG. Results are geometric means⫾SEMs (n⫽6 samples per group per time point). The horizontal dotted lines represent the lower limit of detection.*, Student’sttest,P⬍0.05 compared with KOS. A portion of the control data was previously presented in reference 7.

FIG 2Viral genome loads in latent TG. Mice were infected with 2⫻105_{PFU per eye, and TG were}

collected 28 to 30 days postinfection. DNA was extracted from latent TG, and the amount of HSV-1 DNA present was quantified by real-time PCR (n⫽4 to 10 TG per group). Results shown are the fold reduction compared to KOS. Control data were previously presented in reference 7.

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The efficiency of KOS-NA reactivation is reduced.

To determine if the efficiency

and kinetics of reactivation from latency were altered for KOS-NA, TG of all viral groups

were collected on days 28 to 30 postinfection and subsequently cultured by

explan-tation. Samples were examined daily for cytopathic effect to monitor reactivation, and

the rates of reactivation and total percentage of reactivating samples were determined

for KOS-NA and 7134 mutants relative to that of KOS. KOS began to reactivate on day

3 postexplantation at 66% and reached 100% reactivation by day 5 postexplantation

(Fig. 3). KOS-NA reactivation peaked at day 4 postexplantation, maintaining a

reacti-vation efficiency of 8% throughout the study (Fisher’s exact test,

P

⬍

0.0001) (Fig. 3).

7134 reactivated on day 10 postexplantation, and after heat shock it reached its highest

levels (73%) on day 16 postexplantation (Fig. 3). In contrast, heat shock did not alter the

percentage of TG reactivating KOS-NA. It is very likely that KOS-NA’s diminished

capacity to establish a latent infection (Fig. 2) is directly associated with its inefficient

reactivation phenotype.

Whole-genome sequencing of the KOS-NA mutant.

As our ICP0 phosphorylation

study showed (7), the ICP0 phosphorylation site mutant Phos 3 was not impaired for

acute replication in eyes or TG of mice, the establishment of latency, or reactivation. The

differences in the pathogenesis between Phos 3 and KOS-NA strongly suggested that

KOS-NA contained secondary mutations in its viral genome. To identify the secondary

mutations in KOS-NA responsible for its highly attenuated phenotypes, we sequenced

its viral genome in the same manner as for KOS (8). Subsequently, the genomes of

KOS-NA and KOS (8, 9) were aligned using fast statistical alignment (FSA) to identify

nucleotide polymorphisms between these two viruses (21). The alignment of these

genomes revealed that KOS-NA contained 5 nonsynonymous mutations in three genes:

UL39

, which encodes ICP6,

US7

, which encodes gI, and

RS1

, which encodes the viral

transcriptional regulator ICP4.

Construction of KOS-NAR, UL39

, and HrR3 R viruses.

Because the acute

replication, latency, and explant reactivation capabilities of KOS-NA were remarkably

similar to those of ICP6 mutant viruses in mice (10), we hypothesized that the L393P

(lysine to proline) and/or R950H (arginine to histidine) amino acid substitution encoded

in the open reading frame of ICP6 was responsible, at least in part, for KOS-NA’s

attenuated pathogenesis. To address this possibility, we rescued the UL39 gene of

KOS-NA with a wild-type copy of

UL39

, and we introduced the KOS-NA mutated UL39

gene into the genome of the HrR3 virus, an ICP6::

lacZ

insertion mutant of KOS (18). The

latter HSV-1 mutant we called UL39

. As a control, we also generated an HrR3 rescue

(R) virus in which we rescued the ICP6::

lacZ

cassette with a wild-type copy of

UL39

.

FIG 3Explant-induced reactivation of KOS-NA. Mice were infected with 2⫻105_{PFU per eye. On days 28 to}

30 postinfection, TG were collected and explanted onto Vero cells. The time required for reactivation to occur from the latent TG was determined by assaying the culture medium daily for the presence of infectious virus. Each time point represents the cumulative percentage of samples that showed reactivation (n⫽19 to 20 TG per group). The arrow at the top of the graph indicates that at day 10, samples were heat shocked at 43°C for 3 h. Control data were previously presented in reference 7.

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UL39

mutations are responsible for the diminished acute replication

pheno-type of KOS-NA.

To establish whether the mutations identified in the UL39 gene of

KOS-NA resulted in its attenuated pathogenesis, we tested the acute replication

potential of KOS-NAR and UL39

in the eyes and TG of mice. As shown in Fig. 4A,

UL39

replicated to levels comparable to those of KOS-NA and was reduced up to

500-fold relative to KOS in the eyes of mice at days 1, 3, and 5 postinfection (

t

test,

P

ⱕ

1.2

⫻

10

). In contrast, KOS-NAR and HrR3 R replicated to levels similar to those

of KOS (Fig. 4A).

We then analyzed acute replication in the TG of our viral groups on day 5

postin-fection, as this time point had shown the maximum fold reduction between KOS-NA

and KOS (Fig. 1B). Replication of UL39

in TG at day 5 postinfection could not be

detected (

ⱖ

1,412-fold reduction relative to KOS;

t

test,

P

⫽

6.7

⫻

10

) (Fig. 4B). Acute

replication of KOS-NAR and HrR3 R was comparable to that of KOS (Fig. 4B). The lack of

detectable viral replication for UL39

was also apparent in the TG on day 3

postin-fection (H. H. Mostafa and D. J. Davido, unpublished data).

KOS-NA is reduced for ICP6 protein accumulation without appreciably

affect-ing the interaction between the two subunits of the ribonucleotide reductase.

To

understand the mechanism by which mutations within the UL39 gene impact ICP6, we first

examined the accumulation of ICP6 protein after KOS-NA infection versus KOS. As shown

in Fig. 5A, the ICP6 protein levels after KOS-NA infection were reduced by 50% relative to

FIG 4Mutations in the UL39 gene are responsible for the reduced acute replication phenotype of KOS-NA. (A) Acute replication of KOS-NAR, UL39mut_{, and HrR3 R in mouse eyes. Mice were infected with}

2⫻ 105_{PFU per eye, and tear film was collected from each eye after 4 h and days 1, 3, and 5}

postinfection. The amount of infectious virus collected in each sample was determined by plaque assay. (B) Acute replication of KOS-NAR, UL39mut_{, and HrR3 R in mouse TG at day 5 postinfection. Mice were}

infected as described above, and TG were collected. TG were then homogenized, and the amount of infectious virus present in each sample was determined by plaque assay. Results shown are geometric means_⫾SEMs (n_⫽8 samples per group per time point). The horizontal dotted line represents the lower limit of detection.*, Student’sttest,P⬍0.05.

FIG 5KOS-NA ICP6 protein accumulation and interaction with pUL40. (A) Vero cells were infected at an MOI of 2 with KOS or KOS-NA for 24 h. Infected monolayers were harvested, and the indicated proteins were examined by Western blot analyses. ICP0 was used as a loading control for infection. (B) Vero cells were mock infected (M) or infected at MOIs of 2 for KOS and 5 for KOS-NA for 24 h. UL40 was immunoprecipitated (IP), and the levels of ICP6 and UL40 were analyzed by Western blotting.

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those produced by KOS. On the other hand, the UL40 (small subunit of ribonucleotide

reductase) protein levels produced by the two viruses were comparable (Fig. 5A). This

indicates that one or both substitutions in ICP6 reduce its protein levels. We then examined

the interaction between UL40 and ICP6 produced by KOS-NA versus KOS by

immunopre-cipitating pUL40; however, in these experiments we were unable to pull down detectable

amounts of pUL40 in KOS-NA-infected cells. We also infected cells with KOS-NA at a

mulitiplicity of infection (MOI) higher than for KOS, trying to express more KOS-NA ICP6 and

UL40 proteins. Even under these conditions we could, at best, detect only a very faint UL40

band after immunoprecipitating pUL40 (Fig. 5B). Conversely, a robust amount of pUL40

could be pulled down from KOS-infected cells, and consequently a stronger ICP6 band (Fig.

5B). This result suggests that the conformation of pUL40 may depend on its interaction with

ICP6, and thus, reduced ICP6 levels expressed by KOS-NA might make it difficult to pull

down pUL40 with our antibody. On the other hand, the fact that we were able to detect

some ICP6 when immunoprecipitating pUL40 from KOS-NA-infected cells (Fig. 5B) argues

against the premise that the amino acid substitutions in ICP6 from KOS-NA substantially

affect its interaction with UL40.

Mutation in

UL39

reduces ICP6-mediated inhibition of caspase 8-dependent

apoptosis.

It has been shown that HSV-1 ICP6 binds to caspase 8, inhibiting apoptosis

(15). To test whether the UL39 mutations impaired the antiapoptotic activity of ICP6,

which might be an underlying mechanism of KOS-NA

in vivo

attenuation, we

trans-fected Vero cells with a plasmid expressing human caspase 8 and then intrans-fected these

cells with KOS, KOS-NA, UL39

, or ΔICP6, an ICP6-null mutant. Apoptotic cells were

then counted after staining with Hoechst dye. Our data in Fig. 6 indicate that KOS

impaired caspase 8-induced apoptosis compared to that in KOS-NA-, UL39

-, and

ΔICP6-infected samples. This result indicates that one or both codon changes reduce

the antiapoptotic activity of ICP6.

DISCUSSION

We have isolated a virus, KOS-NA, with two nonsynonymous mutations in the UL39

gene, which encodes the large subunit of ribonucleotide reductase. These mutations

result in amino acid substitutions L393P and R950H. KOS-NA containing these

muta-tions is significantly reduced in its acute replication in mouse corneas and TG, is greatly

impaired in its capacity to establish latency, and poorly reactivates from a latent

infection after explant-induced reactivation.

The HSV-1 ribonucleotide reductase proteins encoded by

UL39

and

UL40

belong to

class I ribonucleotide reductases that have a heterodimeric structure which is active in

the

␣

2

␤

2 configuration (22–26) and is similar to that in mammalian and bacterial

FIG 6UL39mutations from KOS-NA reduce ICP6-mediated inhibition of caspase 8-dependent apoptosis. Vero cells were transfected with a plasmid that expresses either CMV-DsRed (DsRed) or human caspase 8 (C8). Five hours later, Vero cells were treated with acyclovir and after 1 h, cells were infected with each indicated virus at an MOI of 2.5 for 18 h. Cells were stained with Hoechst dye, and apoptotic cells were counted by fluorescence microscopy. Data are the averages from two independent experiments (n⫽6 samples per group). Error bars represent SEMs.*, Student’sttest,P_⬍0.05.

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ribonucleotide reductases. The

UL39

-encoded large subunit contains the catalytic and

allosteric sites which confer substrate specificity. The smaller subunit is responsible for

generation of free radicals that are required for substrate activation (27). Viruses

mutated in the UL39 gene replicate normally in dividing cells at 37°C, where the

reductase activity is thought to be compensated by the cellular homolog. The viral

ribonucleotide reductase activity becomes important, however, for viral DNA

replica-tion in neurons and nondividing cells or at elevated temperatures (e.g., 41°C) (18). Of

relevance to our study, the activity of this viral enzyme complex is essential for efficient

acute replication

in vivo

, which impacts the establishment of latency and reactivation

(10, 28). ICP6 is expressed early, before its small-subunit partner, during viral infection

in cell culture (29–32) and contains an extra N-terminal domain that is not required for

HSV ribonucleotide reductase activity (33). This N-terminal domain possesses

antiapo-ptotic, anti- or pronecroptotic (15–17, 34), chaperone-like (35), and protein kinase (36)

activities. Additionally, ICP6 is a target of HSV-directed cytotoxic T lymphocytes in mice,

a function that is largely conferred by a single amino acid (37).

Previous studies showed that the N-terminal region of ICP6 is required for

interdo-main linking (38), but the interaction of ICP6 with the smaller subunit of ribonucleotide

reductase is conferred by its C-terminal region (39). Amino acid sequence alignment

studies indicate that L393 is not conserved between strain KOS and other HSV strains,

syn17

⫹

(HSV-1), McKrae (HSV-1) HG52 (HSV-2), and 4 other sequenced strains of HSV-1

(Fig. 7A), which, like KOS-NA, encode a proline at this site. This conservation in other

HSV strains suggests that the L393P mutation would not negatively impact ICP6

activities involving this residue and strongly supports the concept that the L393P

substitution in ICP6 of KOS-NA does not contribute to its attenuated phenotype. Amino

acid sequence alignments indicate that R950 is conserved between all HSV-1 and HSV-2

strains we examined (i.e., KOS, syn17

⫹

, McKrae, and the HSV-2 HG52 strain) (Fig. 7B) as

well as varicella-zoster virus (VZV) (40), suggesting that the R950H mutation in KOS-NA

is unique. Moreover, a single mutation at amino acid 961 of HSV-1 ICP6 (41) has been

reported to reduce the ribonucleotide reductase activity of the enzyme (42). Although

this site is important for interactions of the two ribonucleotide reductase subunits, it

was previously concluded that this effect might be indirect (41). Position 950 is close to

amino acid 961, which is within a region identified as “block 10” (residues 960 to 963)

FIG 7Sequence alignments around residue 393 (A) in ICP6 of KOS-NA and various HSV strains and around residue 950 (B) in ICP6 orthologs of KOS-NA, various HSV strains, VZV, and HCMV.

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that is highly conserved between viral ribonucleotide reductases of herpesviruses (40).

Although the region encompassing amino acids 950 and 961 lies in a region highly

conserved between HSV strains, as shown by our sequence alignment (Fig. 7B), this

region was not as highly conserved when we aligned ICP6 to the large ribonucleotide

reductase subunits from human cytomegalovirus (HCMV) and VZV (40) (Fig. 7B) and

other eukaryotic species (data not shown). Nonetheless, the R950 residue itself was

conserved between alpha- and betaherpesviruses we examined and the eukaryotic

large subunits (data not shown). This highlights the functional importance of the R950

site and indicates that it is likely the cause of KOS-NA phenotypes.

Chabaud et al. have shown that there is a constitutive interaction between HSV-1

ICP6 or its HSV-2 homolog, ICP10, and caspase 8 and proposed that this interaction is a

protective mechanism against HSV-induced apoptosis (43). Interestingly, the C-terminal

domain was shown to be essential to inhibit cytokine-induced apoptosis, which is a

conserved activity of ICP10 in HSV-2 (43). In this study, we showed that one amino acid

at the C terminus of HSV-1 ICP6 is responsible for its antiapoptotic effect and its

mutation has deleterious effects on acute viral replication, both in the mouse cornea

and in the TG. The same domain was recently demonstrated to be responsible for ICP6’s

antiapoptotic activity of blocking virus- or cytokine-induced necroptosis (16). Given the

previous studies regarding ICP6 and apoptosis, our data in Fig. 6 strongly suggest that

the H950 residue in ICP6 of KOS-NA impairs caspase 8-induced apoptosis, contributing

to its attenuated phenotype in mice (Fig. 1 to 3). It is not clear if the R950H substitution

in ICP6 of KOS-NA impairs its ribonucleotide reductase activity, although the R950H

substitution does not directly alter conserved motifs known to be required for this

catalytic activity (44).

Deletion of

US7

, which encodes glycoprotein I (gI), can also attenuate the acute viral

replication in sensory ganglia and the central nervous system (CNS) in mice without

having a pronounced effect in cell culture (45, 46). Consequently, we tested the effect

of introducing the KOS-NA

US7

mutation into the genome of ICP0 phosphorylation

mutant Phos 3. Our results showed that this mutation in the background of Phos 3 virus

was able to efficiently replicate in the eyes and TG of mice at levels comparable to those

of the wild-type strain KOS (Mostafa and Davido, unpublished). This result and the

restoration of wild-type replication by KOS-NAR indicate that the

US7

mutation from

KOS-NA is not responsible for the neuroattenuated phenotype of KOS-NA.

The profound attenuation of KOS-NA in the nervous system suggests that this virus

might be useful in safely stimulating immunity to HSV-1. The severe impairment of

KOS-NA in establishing an efficient latent infection and reactivation from latency is

likely related to its diminished acute replication phenotype in the eyes and TG of mice,

as has been observed with other HSV-1 mutants (28, 47). However, KOS-NA injected

directly into the brains of mice also replicates to barely detectable levels (L. A. Morrison

and D. J. Davido, unpublished data). Indeed, we have data from an initial vaccine study

showing that KOS-NA has very good efficacy as a potential prophylactic vaccine against

HSV-1 (D. J. Davido, E. M. Tu, H. Wong, M. Korom, H. H. Mostafa, B. Combs, S. D.

Haenchen, and L. A. Morrison, unpublished data). The efficacy of KOS-NA as a vaccine

may be due, in part, to its ability to express a mutant form of ICP6. Notably, published

studies indicate that expression of HSV-1 ICP6 and its orthologs can generate

ICP6-specific antibodies and cytotoxic T lymphocytes in HSV-infected or vaccinated animals

and individuals (37, 48, 49). These host immune responses would not occur if an

ICP6-null mutant was used as a vaccine.

MATERIALS AND METHODS

Cell lines and viruses.Vero cells and L7 (ICP0-containing Vero) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 ␮g/ml of penicillin, 100 U/ml of streptomycin, and 2 mML-glutamine. The wild-type HSV-1 strain KOS, 7134 (an ICP0-null mutant virus) (20), KOS-NA, KOS-NA marker rescue (MR), UL39mut_{, HrR3 (a virus that contains a}

lacZinsertion inUL39[18]) and ΔICP6 (originally termed ICP6delta [10]), both kindly provided by Sandra Weller, and HrR3 R viruses were propagated and viral titers were determined as previously described (50, 51).

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assembled against the reference HSV-1 strain 17 syn⫹genome using Seqman pro (DNASTAR, Inc.). The final KOS-NA genome is 152,011 bp, has 13 short gaps totaling 1,582 bp exclusively at variable number tandem repeat (VNTR) regions, and was sequenced to an average per-base-pair coverage of 3,104⫻. We transferred annotation from the reference genome strain 17 syn⫹using RATT (56) and confirmed the final KOS-NA annotation manually. To identify and characterize nucleotide differences between KOS and KOS-NA, we aligned the genomes using FSA (21) and applied a custom R script (http://www.r-project .org). We identified 34 single nucleotide polymorphisms (SNPs) discriminating the strains, including 17 nonsynonymous changes residing in six genes (10 of which are the introduced phosphorylation mutations in both copies of ICP0, 2 in the UL39 gene, 4 in both copies of the ICP4 gene, and 1 in the gI gene), and 11 short (1 to 4bp) insertion-deletion events, all present in intergenic regions.

Construction of UL39 mutant and rescue viruses.KOS-NA contains mutations in ICP0 phosphor-ylation sites and was constructed as previously described (7, 51, 52). To generate UL39mut_{, the UL39 gene}

was cloned out of the KOS-NA viral genome using BglII and KpnI sites and ligated into pSP72 using the same sites (pSP72:UL39). Vero cells were plated on 60-mm dishes at 4⫻105_{per plate. Twenty-four hours}

postplating, cells were cotransfected with 1␮g of viral DNA from HrR3 and 2.5␮g of pSP72:UL39 plasmid digested with BglII and KpnI. Transfections were performed using Fugene HD (Roche) at a ratio of 3:1 (microliters of transfection reagent to microgram of DNA) based on manufacturer recommendations. Mutants were identified by blue/white selection in the presence of 5-bromo-4-chloro-3-indolyl-␤-D -galactopyranoside (X-Gal). White plaques were isolated, and viral isolates containing the two mutated sites were screened with the PCR amplification refractory mutation system (ARMS) technique using the following primers for the codon 950 site: WT (R950) primer (5=-CGTGTTTCATCATGCTCTAGC-3=), H950 mutant primer (5=-CGTGTTTCATCATGCTCTAGT-3=), and common primer (5=-TGCACACGGCCTGCCTGAA GCT-3=). Primers for the codon 393 site were WT (L393) primer (5=-CTGGACGTTCCTCCGGTACT-3=), P393 mutant primer (5=-CTGGACGTTCCTCCGGTACC-3=), and common primer (5=-TGGAAGACGGACTCCATGTA G-3=). Candidates were confirmed by DNA sequencing. Correct insertion of the gene into the viral genome was confirmed by XhoI digestion followed by Southern blotting (Mostafa and Davido, unpub-lished). The KOS-NAR virus was generated by cotransfection of 1␮g of KOS-NA viral DNA and 2.5␮g of pKHF plasmid (18), which contains WT UL39, digested with EcoRI and XbaI. Plaques were picked randomly and screened with the PCR ARMS technique as described above. Confirmation of the mutation rescue was performed by PCR, followed by sequencing and Southern blot analyses using the restriction enzyme BamHI (Mostafa and Davido, unpublished). The HrR3 R (rescue) virus was constructed by cotransfecting Vero cells with 1␮g of HrR3 viral DNA and 2.5␮g of pKHF plasmid digested with EcoRI and XbaI. Rescuants were identified by blue/white selection and confirmed by Southern blotting by digesting the viral DNA with XhoI (Mostafa and Davido, unpublished).

Ocular infection of mice.CD-1 outbred female mice (6 to 7 weeks old) were obtained from Charles River Laboratories (Shrewsbury, MA), cared for according toGuide for the Care and Use of Laboratory Animals(57), and infected as previously described (7). Mice were housed according to institutional and federal guidelines and were used in a protocol approved by the University of Kansas. Briefly, mice were anesthetized by intraperitoneal injection of ketamine (75 to 100 mg/kg of body weight) and xylazine (10 mg/kg of body weight). Corneas were scarified with a 26-gauge needle and were infected with KOS, 7134, KOS-NA, KOS-NAR, UL39mut_{, or HrR3 R at 2⫻10}5_{PFU of virus per eye in 3 to 5}␮_{l of medium.}

Determination of viral titers in eyes and TG.Four hours and 1, 3, 5, 7, and/or 9 days postinfection, eye swabs and/or TG samples were collected. For eye swabs, tear film was collected by swabbing the eye with cotton-tipped swabs and placed in microcentrifuge tubes containing 500 ␮l of 5% FBS-supplemented growth medium. For TG samples, the mice were euthanized by CO2asphyxiation, and the

TG were removed and placed in microcentrifuge tubes with 500␮l of growth medium and 100␮l of 1-mm beads. These samples were homogenized using a Mini-Beadbeater 8 (BioSpec, Bartlesville, OK). In all cases, titers of the wild-type, UL39mut_{, and HrR3 R viruses were determined on Vero cells, and titers}

of KOS-NA and KOS-NAR viruses were determined on L7 cells. Statistical analyses were performed using Student’sttest.

Latent viral genome loads in TG.At 28 to 30 days postinfection, latently infected TG were collected, and DNA was isolated from each TG as previously reported (26). PCR primers for the HSV-1 UL50 gene and the mouse adipsin gene were used to amplify viral DNA and as a loading control for cellular DNA, respectively (7). Real-time PCRs were performed in a total volume of 25␮l containing FastStart SYBR green Master (Rox) (Roche, Indianapolis, IN) and primers (300 nM) in an ABI Prism 7500 real-time PCR system (Applied Biosystems, Foster City, CA). UL50 PCR samples contained 125 ng of DNA per reaction

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mixture, adipsin PCR samples contained 10 ng of DNA per reaction mixture, and all samples were analyzed in duplicate or triplicate. Standard curves for each PCR condition were determined as described before (7) to quantify the amount of viral DNA present in each sample relative to the adipsin gene using the threshold cycle (2⫺ΔΔCT_{) method. Statistical analyses were performed using the one-way ANOVA test.} Viral explant-induced reactivation studies.At days 28 to 30 postinfection, latently infected mice were euthanized and each TG was collected, cut into 8 pieces, and cultured on Vero cells in a well of a 24-well plate containing 1.5 ml of growth medium. The cultures were sampled daily for up to 16 days for the presence of infectious virus by cytopathic effect on Vero cells for KOS-infected samples and on L7 cells for KOS-NA- and 7134-infected samples. At day 10 postexplantation, cultures were heat shocked at 43°C for 3 h as an additional stimulus for reactivation. Statistical analyses were performed using Fisher’s exact test.

Western blotting.To examine ICP6 and UL40 protein levels, 1⫻105_{Vero cells were plated per well}

of a 12-well plate. Twenty four hours postplating, cells were infected at an MOI of 2 for each virus. Samples were harvested 24 h postinfection in 50␮l of 1⫻Laemmli buffer (100°C) supplemented with 1⫻ protease inhibitors (leupeptin at 1␮g/ml, aprotinin at 1␮g/ml, and phenylmethylsulfonyl fluoride [PMSF] at 1 mM). Samples were heated at 95°C for 5 min, vortexed, centrifuged, loaded onto a 4 to 12% gradient gel (Invitrogen), and run at 120 V for 1 h. Proteins were transferred to nitrocellulose membranes using a semidry transfer unit (GE Healthcare; catalog no. TE77). Each membrane was blocked in 5% bovine serum albumin in Tris-buffered saline (TBS) and 0.1% Tween 20 (TBS-T) for 1 h at room temperature. Primary antibodies were incubated with membranes overnight at 4°C, then the membranes were washed 3 times in TBS-T, and secondary antibodies were added at room temperature for 1 h. Membranes were washed 3 times in TBS-T and developed using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Pictures were captured with a Kodak 4000R image station.

Immunoprecipitations.Vero cells were plated in 60-mm dishes at 5⫻105_{per plate. Twenty four}

hours later, cells were mock infected or infected with KOS at an MOI of 2 or KOS-NA at MOI of 5 for 24 h. Cells were harvested in 100␮l of a buffer containing 100 mM Tris-HCl (pH 8), 50 mM NaCl, 10% glycerol, 20 mM␤-mercaptoethanol, and 1% Nonidet P-40 with protease inhibitors as described above. Samples were sonicated at 100 W for 30 s, incubated on ice for 30 min, and centrifuged at 15,000 rpm for 10 min at 4°C. After aspiration of the buffer, 50␮l of protein G Dynabeads (Invitrogen) was incubated with 200

␮l of phosphate-buffered saline (PBS) with 0.05% Tween 20 and 5␮l of an anti-UL40 mouse monoclonal antibody (kindly provided by Roger Everett), by rotating for 1 h at room temperature. Beads bound to the antibody were washed with PBS and 0.05% Tween 20 and incubated with the sample lysates, with rocking at 4°C overnight. The beads were then washed twice with PBS-Tween and transferred to new tubes. Fifty microliters of 1⫻Laemmli buffer (100°C) with protease inhibitors was added to each sample, and samples were boiled for 5 min, vortexed, boiled again, and vortexed twice for 1 min each. Western blot analyses were performed as described in the previous section.

Cell viability assay.Vero cells were plated in a 24-well glass plate at 5⫻104_{per well. Twenty-four}

hours later, cells were transfected with 1.25␮g of a plasmid that expresses either CMV-DsRed (gift from Yoshi Azuma) or human caspase 8 (pcDNA3-Casp8) (Addgene; plasmid 11817) (58). Transfections were performed using Opti-MEM (Invitrogen) and Fugene HD (Promega). Five hours after transfection, medium was removed and Vero cell medium containing acyclovir (ACV; 44␮g/ml) was added to inhibit viral DNA replication. Six hours after transfection, cells were infected with each virus at an MOI of 2.5 for 1 h in the presence of ACV and washed three times with PBS plus ACV; Vero cell medium containing ACV was then added to the cells. Twenty-four hours after transfection, cells were stained with Hoechst 33342 dye (Invitrogen) by adding 0.5␮l of 10-mg/ml aqueous dye stock solution to 0.5 ml of phenol red-free DMEM (Thermo Scientific) in each well. Apoptotic cells were identified as having condensed chromatin/ fragmented nuclei compared to the total number of cells per field of vision by fluorescence microscopy. Three independent sets of images were taken for each sample. Statistical analyses were performed using Student’s t test (GraphPad Prism) for virally infected samples compared to caspase 8-transfected, KOS-infected cells.

ACKNOWLEDGMENTS

This work was supported by University of Kansas (D.J.D.) and National Institutes of

Health (NIH) grants R21EY019739 (L.A.M. and D.J.D.) and R01OD010974 (S.J.M.). The

Genome Technology Access Center is partially supported by NCI Cancer Center Support

grant no. P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA grant no.

UL1RR024992 from the National Center for Research Resources (NCRR), a component of

the NIH, and by the NIH Roadmap for Medical Research.

D.J.D. and L.A.M. are coinventors on U.S. patent 9616119, which describes using

KOS-NA as a vaccine against HSV-1 infections.

We thank Sandra Weller, Howard Marsden, and Roger Everett for providing reagents

related to this study.

The content of this article is solely the responsibility of the authors and does not

necessarily represent the official views of the NIH.

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