FULL PAPER
Virology
Role of the UL41 Protein of Pseudorabies Virus in Host Shutoff, Pathogenesis and
Induction of TNF- Expression
Hui-Wen LIN1), Wei-Li HSU2), Yuan-Yen CHANG3,4), Ming-Shiou JAN5), Min-Liang WONG1) and Tien-Jye CHANG1)*
1)
Department of Veterinary Medicine and 2)Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine,
National Chung-Hsing University, Taichung, 3)Department of Microbiology and Immunology, School of Medicine, Chung Shan Medical
University, Taichung, 4)Clinical Laboratory, Chung Shan Medical University Hospital, Taichung and 5)Institute of Immunology, School of
Medicine, Chung Shan Medical University, Taichung, Taiwan
(Received 9 February 2010/Accepted 10 April 2010/Published online in J-STAGE 24 April 2010)
ABSTRACT.
The vhs (virion host shutoff) is highly conserved in alphaherpesvirus, including pseudorabies virus (PRV). In an attempt to
explore the function of vhs of PRV, we constructed and characterized a mutant virus (41). In the absence of vhs activity, 41 mutant
is highly attenuated in mice model and the lethality is correlated with the virus dissemination in neural tissues. As with herpes simplex
virus type 1 (HSV-1), the prototype virus of alphaherpesvirus, the pronounced decrease in cellular protein synthesis triggered by wild
type PRV was largely restored in cells infected with 41 virus. Furthermore, tumor necrosis factor- (TNF-) protein expression was
elevated significantly in spleen of mice infected with vhs mutant virus. Since TNF- has been indicated to be an important cytokine in
the innate immune response against various infections, our results implicate vhs may contribute to the protection against PRV lethality
via the action of TNF-. Overall, we confirm the shutoff function of vhs protein in PRV, and demonstrate the role that vhs protein plays
in virulence, and regulation of cytokine production.
KEY WORDS: pseudorabies virus (PRV), tumor necrosis factor- (TNF-), UL41, virion host shutoff (vhs) protein.
J. Vet. Med. Sci. 72(9): 1179–1187, 2010
Pseudorabies virus (PRV) is a swine herpesvirus belonging to the Alphaherpesvirinae, the best characterized virus
of this family being herpes simplex virus type 1 (HSV-1)
[21]. One of the hallmarks of productive HSV-1 infection is
the shutoff of host protein synthesis that helps redirect the
cellular translation machinery to viral protein synthesis.
The decrease in host protein synthesis is achieved mainly
through the actions of the virion host shutoff (vhs) protein
encoded by UL41 gene and ICP27 [10, 11]. Earlier reports
showed that vhs harbors mRNA-specific ribonuclease activities via disruption of preexisting polyribosomes leading to
the shutoff of protein synthesis [8, 22, 24]. Very recently, a
number of cellular mRNAs have been demonstrated to be
the targets of vhs [3–5, 29]. Not only destabilizing cellular
mRNA, the ribonuclease activity of vhs also accelerates the
turnover of viral mRNAs which is hypothesized to facilitate
the sequential expression of the three kinetic classes of viral
genes [16, 27].
The endonuclease activity of vhs has been demonstrated
with purified protein expressed in prokaryotic system [30].
Recently, selective targeting of vhs was observed in several
distinct sequence contexts. It was demonstrated that vhs
specifically destabilizes regions of AU-rich elements at the
3’ end of IEX-1 mRNA [30], although the mechanism of
IEX-1 transcript decay in the course of HSV-1 infection
remains unclear [4, 13]. Several lines of evidence have indicated a role for vhs protein in the regulation of the immune
response. Gopinath et al. revealed that down-regulation of
* CORRESPONDENCE TO: CHANG, T.-J., Department of Veterinary
Medicine, National Chung-Hsing University, 250 Kuo Kuang
Road, Taichung 40227, Taiwan.
e-mail: [email protected]
MHC class I expression by bovine herpesvirus 1 is mediated
by vhs activity [9, 15]. Furthermore, the production of several proinflammatory chemokines and cytokines, including
interleukin-1 (IL-1), IL-8 and MIP-1, were suppressed
by vhs in U937 cells and in HEL cells infected with HSV1[28]. These observations suggest that vhs may have a role
in pathogenesis through regulation of the anti-viral
response.
The PRV vhs protein, encoded by UL41, consists of 365
amino acids which make it shorter than its homologue in
HSV-1 (489 aa) [19]. While sequence analysis reveals that
the amino acid identity is approximately 38.4% compared to
that of HSV-1 UL41, four conserved functional domains
still remain in PRV vhs [1]. Our previous study has shown
that purified vhs protein expressed in E. coli displays ribonuclease activity in in vitro assays [17]. While the roles that
vhs plays in the HSV-1 viral life cycle, latency and pathogenesis have been extensively studied [26], little is known
about the contribution of vhs in PRV infection; hence, the
goal of this study was to investigate the role of vhs during
PRV infection. More specifically, we asked whether vhs is
required for triggering virion host shutoff. Here, we generated and characterized a vhs null mutant (41) and compared its virion host shutoff activity with that of wild type
PRV (TNL strain). In addition, we investigated the effects
of UL41 on PRV virulence in mice, as well as the stability
of cytokine transcripts and production of related proteins.
MATERIALS AND METHODS.
Cell culture and virus: Porcine kidney 15 (PK 15) cells
were maintained in Dulbecco’s modified Eagle’s medium
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H-W. LIN ET AL.
(DMEM; Hyclone, Logan, Utah, U.S.A.) supplemented
with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah).
Rabbit kidney (RK 13) cells were maintained in Eagle’s
minimal essential medium (MEM) supplemented with 10%
FBS. RAW264.7 cell line was cultured in RPMI1640
(Hyclone, Logan, Utah) supplemented with 10% FBS. The
stock of wild type (wt) PRV (strain TNL) and the vhs
mutant virus (41) used in this study were amplified from
PK15 cells and the titer was determined by standard plaque
assay in PK15 cells [17].
Plasmids: To simplify vhs deletion mutant virus screening, the coding region of UL41 was replaced by that of green
fluorescence protein (GFP). To do so, plasmids containing
GFP, UL40 and UL41 genes were initially constructed. A
plasmid harboring a partial UL42 gene (pGEM-T-vhsp) was
made as described previously [2]. The GFP coding region
obtained from vector pEGFP-N1 (CLONTECH, Palo Alto,
CA, U.S.A.) was inserted into pcDNA3.1(–) at the restriction sites Bam HI and Pvu II, and the resulting plasmid was
named 3.1-EGFP. The viral UL40 gene was amplified from
the genome of PRV TNL strain by PCR using the primers
UL40-F (5’-GCGCCATGGAGTACTTTTACA-3’) and
UL40-R (5’-GCTAGTGTCTCAACGCTTTAT-3’). The
PCR product was cloned into pGEM-T-Easy vector, and the
resulting plasmid was named pGEM-T-Z-UL40. Subsequently, we constructed the plasmid p41-EGFP, in which
entire vhs open reading frame was replaced by the GFP coding region flanked with UL40 and UL42 genes at either end.
The UL41 deletion cassette was constructed using several
steps: firstly, the EGFP coding region from 3.1-EGFP was
inserted downstream of UL42 in pGEM-T-vhsp digested
with Eag I and Spe I, resulting in the plasmid vhsp-EGFP.
Subsequently, the UL40 gene was isolated from plasmid
pGEM-T-Z-UL40 and inserted in vhsp-EGFP at the Not I
restriction site to generate plasmid vhsp-EGFP-UL40 in
which the expression of vhs was under control of its authentic promoter. In addition, for ease of plaque purification we
constructed a vhs deletion cassette (p41-EGFP) in which
GFP expression was driven by the potent CMV (cytomegalovirus) promoter. To do so, a Bgl II and Age I digested
fragment containing the CMV promoter obtained from 3.1EGFP was inserted upstream of GPF in plasmid vhspEGFP-UL40 cut with Bam HI and Age I. The corresponding
positions of the UL41 gene and the recombinant GFP
expression cassette within the PRV genome are schematically illustrated in Fig. 1A.
Generation of a vhs-deletion PRV virus: pGEM-TUL41GFP plasmid was linearized with Msc I and cotransfected with PRV-TNL DNA into PK15 cells using Lipofectamine transfection reagent (Invitrogen, Carlsbad,
California, U.S.A.). The transfected cells were cultured in
DMEM containing 1% methylcellulose. Green fluorescent
plaques were picked under a fluorescent microscope, and
progeny strains were enriched and purified to homogeneity
by three rounds of plaque purification.
Purification of wt PRV and 41 DNA: PK15 cells were
infected with PRV wt or 41 at an MOI of 0.5. Infected
cells were harvested with lysis buffer (Sigma Co.,
Ronkonkoma, NY, U.S.A.; 100 mM NaCl, 10 mM TrisHCl, (pH 8.0), 25 mM EDTA, 0.5% SDS, 0.25 mg/ml proteinase K) and incubated at 37C for 2 hr. The total DNA
was purified by phenol-extraction and precipitated with two
volumes of 100% ethanol and 1/10th volume 3 M sodium
acetate for 30 min at –20C. After centrifugation, DNA pellets were resuspended in TE buffer (Sigma Co; 10 mM Tris
(pH 8.0), 1 mM EDTA).
Southern blot analysis of vhs deletion mutant virus: Ten
g genomic DNA from PRV TNL or 41 were digested
with Xho I, separated on a 1% agarose gel and transferred to
nylon membrane for southern blot analysis. The EGFP
gene, prepared from 3.1-EGFP plasmid digested by Mlu I,
was used as a probe. Probe of the UL41 gene was prepared
from pGEM-T-vhsFN/Rd (unpublished data) plasmid
digested by Nco I. The UL40 gene was prepared from
pGEM-T-UL40 plasmid digested by Puv II. Probes for
southern blotting were prepared using a DIG RNA Labeling
Kit according to the manufacturer’s directions (Roche, Bedford, MA, U.S.A.).
Analysis of growth kinetics: To monitor kinetics of viral
replication, PK15 cells were infected with PRV at MOIs of
0.01, 0.1, and 1. Following a one hr adsorption at 37C, the
medium was removed and replaced with fresh growth
medium. At indicated times, the cells were harvested by
three freeze-thaw cycles, and the virus titer was determined
by standard plaque assay.
Protein synthesis assay: RK-13 cells (4.7  106 cells per
60 mm diameter petri dish) were infected at 1 PFU per cell
for 1 hr at 37C. At the indicated infection time points, the
cells were washed three times with cold 1X PBS and incubated with DMEM without methionine at 37C for 30 min.
Subsequently, the medium was replaced with 1 ml of
DMEM (without methionine) containing 50 þCi [35S], and
the cells were incubated at 37C. After 1 hr, cells were
washed with cold 1X PBS, and cell pellets were collected in
200 l 2X sample buffer. Cell extracts were boiled for 5
min and resolved by 10% SDS-PAGE at 70 V overnight.
The gel was then dried and autoradiographed.
Northern blot analysis: PK15 cells were either mocktreated or infected with wt PRV or vhs mutant at an MOI of
10. At various time points after infection, total RNA was
isolated using a RareRNA kit (GENEPURE, Taipei, Taiwan). The RNA (10 g) was electrophoresed on a 1.5%
RNA denaturing agarose gel and transferred to a nylon
membrane using standard upward capillary transfer overnight. Prehybridization and hybridization were done with
the ExpressHyb buffer (Clontech, Palo Alto, CA, U.S.A.).
The filter was probed for swine -actin. The membranes
were washed using protocols suggested by the manufacturer
and exposed to X-film for signal detection. Signals were
quantified with the Alpha Imager 2000 documentation and
analysis system. The level of -actin for mock-infected
cells was arbitrarily set as 100%, and -actin values of cell
infected with wt- or 41- PRV were normalized to mock
values.
ROLE OF THE UL41 PROTEIN OF PSEUDORABIES VIRUS
Infection of mice: Six- to eight-week-old mice of BABL/
c were used for all experiments. To determine mean survival times, five mice in each group were inoculated with
PRV wt or 41 viruses. Ten microliters of the respective
virus suspension containing 106 PFU was intranasally
instilled into mice. Animals were observed twice a day.
Recovery of infectious virus from brain and trigeminal
ganglia (TG): Three mice for each time point (24, 48 hpi)
were anesthetized with 200 l of urethane (1,500 mg/kg).
Brain and TG were dissected from the mice; half of brain or
TG was weighed and kept in 500 or 200  l of DMEM
medium with 2% FBS, respectively. Subsequently, samples
were homogenized by pestle followed by two flash frozenthaw cycles. The virus titers in the respective tissues were
determined with standard plaque assay in PK15 cells [17].
Isolation and propagation of spleen cells: Whole spleens
of infected mice were aseptically removed from mice and
teased on a steel mesh in chilled RPMI1640 medium in a
plastic dish. The cells passed through were then washed
twice with the medium. The nucleated cells were washed
twice and resuspended in RPMI-1640 medium supplemented with 10 % FBS. 3.2  106 spleen cells were placed
in each well of a 6-well plate in 3 ml of complete medium,
supplemented with 10% RPMI1640. After 48 hr, the cultures medium were harvested for ELISA assay. Expression
of TNF- was measured by ELISA kit according to the
manufacturer’s instructions (eBioscience, San Diego, CA,
U.S.A.). All the experiments were done in triplicates.
Determination of TNF- secreted from RAW264.7 cells:
The media of RAW264.7 cells either mock infected or
infected with wt PRV or ¢41 at MOI of 0.2, 1 and 5 were
harvested at the time indicated. The amount of TNF- in
supernatant was detected for by ELISA kit (eBioscience San
Diego, CA).
RESULTS
Construction of the vhs-deletion mutant PRV: In order to
understand the function of vhs protein in detail, we generated a vhs-deficient mutant PRV (41), in which the entire
coding region of UL41 was replaced by GFP gene. As
expected, 41plaque with high fluorescence intensity was
used for further characterization.
Genotype analysis of the recombinant viruses: Southern
blot analysis was performed to analyze the genotype and the
region of recombination. The genomic structure of wt and
41 is illustrated in Fig. 1A, and the location of probes for
detection of UL41, EGFP and UL40 is shown in Fig. 1B.
Viral genomic DNA probed with UL40 gave the expected
Xho I- fragments sizes of 2.5 kb and 2.0 kb for wt PRV, but
fragments of smaller size (1.7 kb and 2.0 kb) were detected
in the 41 virus (Fig. 1C). As expected, a probe harboring
vhs coding sequences did not hybridize with the 41 mutant
virus (lane 2), while the EGFP-specific probe did not recognize wt PRV (lane 1 in Fig. 1C).
Growth curve and Effect of the vhs-deletion mutant upon
cellular protein synthesis: Growth of the vhs null mutant
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virus (41) was monitored and compared with that of wt
PRV. Progeny yield of wt PRV and 41 in PK-15 cells was
measured at 12, 24 and 36 hr post-infection (hpi). With an
MOI of 1, the virus yield of 41 was approximately 3-5 fold
lower than that of wt PRV at all time points (Fig. 2). Similar
results were observed in cells infected with lower virus
inoculums (MOI of 0.1 or 0.01). Although there was only a
moderate decrease in progeny yield, the plaque size of 41
was significantly reduced compared to that of wt PRV (Fig.
2). To explore the potential role of vhs on host protein synthesis during PRV infection, we analyzed the global protein
expression profile of infected RK-13 cells, which is highly
susceptible to PRV, by a metabolic labeling assay. As indicated with asterisks in Fig. 3, expression of several viral proteins were observed in the course of infection; more
specifically at early time points of infection (1 to 3 hr post
absorption), e.g. the viral protein indicated with #. Noticeably, the pattern of protein synthesis in wt PRV infected
cells was accompanied by a reduction of several major proteins with molecular weights ranging from 110 to 35 kDa,
indicated by arrows. Over all, the shutoff activity triggered
by wt PRV was remarkably reduced in the absence of vhs
activity (Fig. 3). Consistently, the shutoff of host protein
synthesis results was also observed in PK cells infected with
41 virus, but to a lesser extent (data not shown). The
apparent shutoff of cellular protein synthesis at 7 hpa
observed in cells infected with the vhs mutant was mostly
due to the pipetting error, as evidenced by a global decrease
of protein synthesis including the viral proteins. One might
argue that the reduced shutoff function observed in 41
virus may result from the lower efficiency of vhs mutant
virus replication compared to wt PRV. Nevertheless, as
shown in Fig. 3 most of viral proteins, especially those with
high molecular weights indicated with asterisks, were synthesized equivalently well, suggesting that vhs mutant virus
replicates at a similar rate as does wild type virus.
Contribution of vhs to the RNA accumulation during PRV
infection: Many research groups have reported that the shutoff of protein synthesis is mediated by the ribonuclease
(RNase) activity of vhs at early stages in the HSV replication cycle [7, 12]. However, the role that vhs plays in RNA
accumulation during PRV infection remains unclear. To
address this question, Northern blot analysis was performed.
As illustrated in Fig. 4A. a pronounced decrease of -actin
mRNA accumulation was detected as early as 3 hpi, and a
progressive degradation was observed in cells infected with
wt PRV. By contrast, in the absence of vhs protein, the
onset of -actin mRNA degradation was slower and proceeded to a much lesser extent. Indeed, the most significant
degradation of -actin mRNA occurred at 9 hpi during 41
infection. At this time point, 70% of the signal present in
mock infected cells remained, which is significantly more
than that in wt PRV infected cells (30%) (Fig. 4B). Hence,
our results demonstrate that infection of PRV triggers RNA
degradation and that the RNase activity of vhs may contribute to the shutoff phenomenon.
41 virus was attenuated in mice model of PRV infection:
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Fig. 1. Schematic illustration of the constructs and Southern blot analysis of the UL41 deletion mutant
(41). The Xho I fragment from the UL region of the viral genome harbors UL41, the gene encoding
vhs, and neighboring genes such as UL42, UL40 and UL39. In the construct for the vhs null mutant,
the UL41 region was replaced by an expression cassette consisting of the EGFP gene (A). The location of probes used for Southern blot is indicated (B). Viral genomic DNA of wild type (wt; lane 1) or
41 virus (lane 2) digested with Xho I was separated by agarose gel electrophoresis and transferred to
a nylon membrane. The blot was hybridized with DIG-labelled probes specific for UL40, UL41 and
EGFP genes (C). Molecular sizes are indicated at the left of each panel.
Several lines of evidence have shown that shutoff activity of
vhs being associated with an anti-viral mechanism in the
course of HSV infection [28]. Consistent with HSV-1,
despite the similar growth kinetics with wild-type virus, a
considerable prolonged survival time and delay onset of
symptoms was observed in mice infected with 41 virus.
Mice infected with wild type PRV, severe symptoms of
infection was developed as early as 48 hpi and 100% mortality was observed at day 3; in contrast, no death occurred in
the mice infected with 41 virus (100% survived). The survival rate of mice infected with 41 virus as 80% at day 4
(Fig. 5A).
Replication in mice brain and trigeminal ganglian (TG):
To explore whether the highly attenuation of 41 in mice is
due to the restraint of virus replication, the growth of PRV
wt and 41 mutant virus in mouse brain and TG in acute
replication was examined. As shown in Fig. 5, titer of 1.7 
104 and 1  103 pfu/ml of PRV wt was amplified in TG (Fig.
5B) and brain (Fig. 5C) respectively. Surprisingly, titers of
41 were under detection in both tissues. Considering the
mice resistant to lethal infection of vhs mutant virus, lower
virulence of 41 mutant virus seems to correlate with the
ROLE OF THE UL41 PROTEIN OF PSEUDORABIES VIRUS
Fig. 2. Growth kinetics of PRV (wt) and vhs mutant virus (41).
(A) PK15 cells were infected with PRV wt or 41 at an MOI of 1.
After 1.5 hr inoculation, virus inoculums were removed and
replaced with fresh growth medium. The infections were halted at
12, 24, and 36 hpi. After three freeze/thaw cycles, virus yields
were determined on PK15 cells. Each time point was performed
in triplicate by a standard plaque assay. The data represent the
average number of PFU/ml.
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delay of virus spreading in the nerve tissues. These observations coincide with what were concluded by Leib’s in HSV
infection [26]. Overall, these results indicate 41 mutant
virus displays a marked attenuation of virulence in a mouse
model.
Quantification of TNF-  expression in PRV or  41
infected spleen cells: Cantin and coworkers demonstrated
that deficient of TNF- activity results in significantly
increased susceptibility to fatal HSV encephalitis (HSVE)
in C57BL/6 mice and prolonged persistence of elevated levels of virus in neural tissues [18]. To understand whether
TNF- contributes to the basis for this reduced pathogenicity of these 2 PRV viruses in mice, we compared that the
TNF- expression level in the spleen cells isolated from
mice infected with wt PRV and the vhs mutant virus 41. In
wild type virus infection, expression of TNF- were upregulated (~ 50 pg/ml) till 24 hpi followed by declined to under
detection level at 48 hpi (Fig. 6A). In contrast, a significant
increase of TNF- (~150 pg/ml) expression was observed in
spleen cells isolated from 41 infected mice at 48 hpi. Similar results were shown in an in vitro model: As demonstrated in Fig. 6B, there was an approximate 4- to 5-fold
increase in TNF- protein detected in RAW264.7cells,
mouse macrophage-like cells, infected with 41 at an MOI
of 5 as compared to a wt infection. Similar results were
Fig. 3. Shutoff of host protein synthesis in cells infected with wt PRV and 41 virus. RK13
cells were mock-infected or infected with PRV wt or 41 at an MOI of 1. At 0, 1, 3, 5, 7
hr post absorption (hpa), the infected cells were metabolically labeled with 50 Ci [35S]
for 1 hr. Whole cell lysate was resolved by 10% SDS-PAGE, and the profile of protein
synthesis was revealed by radiography. In general, the progression of shutoff of protein
synthesis observed in wild type PRV infected cells was slowed down in vhs mutant
infected cells; as indicated with arrows. Several viral proteins expressed in high level
were observed, especially those with high molecular weights indicated with asterisks.
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Fig. 4. Effect of vhs mutant virus on accumulation of -actin mRNA in infected cells.
PK15 cells were either mock-infected, or infected with PRV wt or 41 at an MOI of 10,
and total RNA was harvested at 0, 3, 6, 9 hpi. (A) For northern blot analysis, 10 g of
RNA was resolved in 1% agarose gel (lower panel) and 32P-labeled DNA probe was prepared for the detection of -actin RNA. (B) The Intensity of -actin was quantified by
phosphorimage analysis and the decrease in -actin mRNA accumulation in infected
cells was then evaluated by normalization with that in mock-infected. The data are the
average of three independent Northern blotting experiments and the error bars indicates
the standard deviations from three repeats.
observed at MOI of 1 (Fig. 6C) and MOI of 0.2 (data not
shown). Accumulation of TNF- RNA in PRV infected
cells was also evaluated by semi-quantitative RT-PCR. At
early stages of wt PRV infection, the expression level of
TNF- was similar to that in uninfected cells; however,
expression was significantly induced by 5 hpi (data not
shown). Consistently, a pronounced induction of TNF-
was observed in cells infected with 41 virus at 1 and 3 hpi,
but this induction declined after 3 hpi (data not shown).
DISCUSSION
Although Klopfleisch et al., has previously demonstrated
that a PRV vhs deletion mutant (a mutant lacking codons 21
to 349 of the vhs) had light to moderate influence on neuroinvasion and neurovirulence [14] , this is the first study of
characterization of the host shutoff phenomenon by vhs in
PRV infected cells. In present study, the role of UL41
encoded vhs protein in pseudorabies virus (PRV, TNL
strain) replication was characterized using a vhs loss of
function mutant (41) in which the entire coding region of
vhs was replaced by a GFP expression cassette under the
control of a CMV promoter. Similar to previous observations recorded for HSV-1, the vhs mutant virus 41 shared
similar growth kinetics with its wild type (wt) counterpart,
and the yield of 41 progeny was only reduced by 3- to 5fold compared to wt PRV at all infection times analyzed [25,
26]. These results indicate that the function of vhs may be
dispensable for PRV replication in cell culture systems. The
hallmark of herpes virus infection is the shutoff of host protein synthesis, and the UL41 gene product (vhs) seems
responsible for this activity. Although UL41 is well conserved among alphaherpesviruses, the virion shutoff activity may vary considerably among different virus stains. For
instance, an early study demonstrated that EHV-1 was incapable of triggering the virion-associated shutoff of cellular
protein synthesis, and no vhs-induced cellular mRNA degradation was detected [6]. Hence, it is important that the
ROLE OF THE UL41 PROTEIN OF PSEUDORABIES VIRUS
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Fig. 5. 41 virus is highly attenuated in BABL/c mice. BABL/c mice were inoculated with 106 PFU of PRV wt or 41 by intranasal
route and survival was assessed daily (A). The low virulence is associated with the delay of virus spreading in the nerve tissues. Mice
were anesthetized at the indicated times after intranasal inoculation with 106 PFU of wt or 41 PRV viruses. Mice TG (B) and brain (C)
were removed, processed, and assayed for viral loads by a standard plaque assay. The same experiments were repeated three times.
function of vhs protein in PRV replication be investigated in
more detail. Our results from metabolic labeling experiments demonstrate that the strong inhibition of host protein
synthesis in wt PRV infected RK-13 cells coincides with the
presence of vhs, indicating that the UL41 gene product contributes to the host shutoff phenomenon. As vhs is a tegument protein, which is synthesized at later time points of
infection, early shutoff effects observed in Fig. 3 are likely
due to vhs delivered by virions or/and by other early
expressed viral protein. In HSV-1, the decrease in host protein synthesis is achieved through the actions of the vhs, and
ICP27, an immediate early protein [10, 11]. However,
whether other protein involved in PRV virion host shutoff
remains unclear.
Degradation of  -actin mRNA was obtained in cells
infected with wt PRV but not in the vhs null mutant infected
cells (Fig. 4), indicating that ribonuclease activity of vhs
protein is very likely related to its shutoff activity. One
might argue that the reduced shutoff function observed in
41 virus may result from the lower efficiency of vhs
mutant virus replication compared to wt PRV. Nevertheless, as shown in Fig. 3 most of viral proteins, especially
those with high molecular weights indicated with asterisks,
were synthesized equivalently well in PK15 cells, suggesting that vhs mutant virus replicates at a similar rate as does
wild type virus. In addition, a wild type PRV harboring
GFP coding region, inserted at Us9 and Us2 regions, was
included as parallel control in experiments of viral growth
kinetics and mice infection. Results revealed GFP-wt PRV
behaves almost indistinguishable from that of the authentic
wt PRV (data not shown), which indicates expression of
GFP unlikely has impact on PRV replication and it’s cellu-
lar effect.
Several lines of evidence have shown that shutoff activity
of vhs being associated with an anti-viral mechanism in the
course of HSV infection. With respect to virulence, we
found that when BALB/c mice were intranasally challenged
with either wt PRV or 41 virus at a dosage of 1  106 PFU
per mouse (5 mice per group), while 100% of wt PRV
infected mice died within 3 days, 80% of 41 infected mice
survived to day 5 until sacrificed (Fig. 5A). The prolong
survival time of vhs mutant PRV was also demonstrated by
Mettenleiter’s research group done with different mice species [14]. Mice resistant to lethal infection of vhs mutant
virus seems to correlate with the delay of virus spreading in
the nerve tissues as evidenced by the analysis of virus loads
in the trigeminal ganglion and brain. While wt virus was
readily detectable (4.5  102 PFU/ml) on 24 hpi in TG;
increased virus tilter i.e. 1.7  104, and 1.1  103 PFU/ml in
TG and in brain, respectively, were obtained on 48 hpi, replication of 41 was completely abolished in both trigeminal
ganglia, and in brains of mice. These observations coincide
with what were concluded in HSV infection [26]. Overall,
these results indicate despite the similar growth kinetics
observed in an in vitro system, 41 mutant virus displays a
marked attenuation of virulence in a mouse model.
Accumulating data have indicated the role of vhs play in
virulence may associate with targeting host innate immune
response via it’s ribonuclease activity [28]. Suzutani et al.
demonstrated that vhs participates in the evasion of non-specific host immunity via degradation of several cytokine
mRNA for instance type I IFN, IL-8 during HSV-1 infection
[28]. In this study, a significant elevated TNF- expression
(150 pg/ml) and a 3-4 fold increase of TNF- protein secre-
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H-W. LIN ET AL.
Fig. 6. PRV infection induced TNF- expression in mice model and cell culture. TNF- level were measured in spleen cells obtained from mice intranasally infected with 106 PFU of wt or 41 PRV viruses at 24 or 48 hpi as
indicated (A). Expression of TNF- was also analyzed in cell culture system.
RAW264.7 cells were either mock infected ,or infected with PRV wt or 41
viruses at MOI of 5 (B) or 1 (C). At 0, 3, 6, 12, and 24 hpi, the supernatant of
infected cells was collected and the expression of TNF- protein was quantified by ELISA. The data are the average of three independent experiments and
the error bars indicates the standard deviations from three repeats.
tion were detected in spleen cells and in macrophage-like
cells infected with 41 virus, compared with wild type
PRV. TNF- has been shown to be an important cytokine in
the innate immune response against various infections,
including HSV. In acute infection, the lowest survival rate
was observed in TNF- knock out mice infected with HSV1 via the corneal rout [18, 20]. In an encephalitis mouse
model, HSV-1 was lethal to TNF- deficient mice, and the
brain viral loads were higher than that in wild type mice,
indicating that TNF- is a crucial factor in protection
against HSV-1 infection [23]. Taken together, these data
show that PRV (TNL strain) defective in vhs function
exhibits a reduced activity in the shut-down of host protein
synthesis. Furthermore, the virulence of PRV was greatly
attenuated in the absence of vhs activity, and virus replication of 41was failed to be detected in mice brain as well as
TG, indicating that the vhs mediated RNA degradation
might be one of the strategies that PRV employs to inactivate host functions detrimental to its replication and spread.
We showed the expression of TNF- protein increased significantly in vhs mutant viruses. Whether TNF- directly
contributes to non-specific host defense mechanisms of this
mutant requires further investigation.
ACKNOWLEDGMENTS. This work was supported by the
National Science Council (grant number: NSC96-2313-B005-016-MY3) and in part by the Ministry of Education,
Taiwan, under the ATU plan. The animal protocol used in
this study has been approved by Institutional Animal Care
and Use Committee (IACUC Approval No. 95-62). The
authors wish to thank Ms. Holly Saffran, Department of
Medical Microbiology and Immunology, University of
ROLE OF THE UL41 PROTEIN OF PSEUDORABIES VIRUS
Alberta, Edmonton, Alberta, Canada, for editorial assistance.
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