Department of Microbiology, Asahikawa Medical College, Asahikawa 078-8510, Japan1
Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan2
Author for correspondence: Tatsuo Suzutani. Fax +81 166 68 2399. e-mail suzutani{at}asahikawa-med.ac.jp
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Abstract |
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Introduction |
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The UL41 gene is an early/late gene and expresses two forms of the protein, the major 58 kDa polypeptide and a highly phosphorylated 59·5 kDa polypeptide (Read et al., 1993 ). At the intermediate and late stages of the HSV replication cycle, the shutoff activity of the newly synthesized vhs polypeptide is suppressed by formation of a complex with VP16 (also known as Vmw65,
TIF and ICP25) via a domain spanning residues 310330 of the vhs protein (Lam et al., 1996
; Schmelter et al., 1996
). The 58 kDa vhsVP16 complex is packaged into the tegument of HSV particles, and delivered into the cytoplasm of newly infected cells following fusion of the viral envelope with the host plasma membrane (McLauchlan et al., 1992
; Smibert et al., 1992
; Read et al., 1993
). UL13 protein kinase seems to be involved in releasing vhs protein and VP16 from the tegument, although the details have yet to be precisely defined (Overton et al., 1994
). The vhs protein degrades both cellular and viral mRNAs rapidly, resulting in the shutoff of protein synthesis, while it does not affect rRNA or tRNA (Nishioka & Silverstein, 1977
; Schek & Bachenheimer, 1985
; Strom & Frenkel, 1987
). Zelus et al. (1996)
demonstrated a messenger ribonuclease (RNase) activity in crude vhs samples which were prepared from purified virions or synthesized in a rabbit reticulocyte in vitro translation system. This study strongly suggested that vhs protein itself was an RNase, although a demonstration of the RNase activity of vhs protein purified to homogeneity is necessary.
The role of vhs function in virus replication has been studied using vhs loss of function mutants. The studies indicate that: (i) shutoff of expression of host genes may allow better utilization of the translational machinery of the infected cell for translation of newly synthesized viral mRNAs, (ii) degradation of viral mRNA may facilitate rapid transitions in the expression of specific groups of viral genes (Kwong & Frenkel, 1987 ; Strom & Frenkel, 1987
). However, these functions are dispensable, and loss of vhs function reduces virus yield by less than 1 log order in tissue culture infection (Read & Frenkel, 1983
; Strelow & Leib, 1995
). The vhs protein seems to play more important role(s) in vivo, as indicated by the reduced virulence and the impaired replication of vhs mutants in mouse (Becker et al., 1993
; Strelow & Leib, 1995
). Therefore, we have studied the molecular mechanism of the low virulence of vhs-deficient mutants to understand how the degradation of host mRNA is linked to virulence. The results presented here indicate that vhs function inhibits non-specific host defence mechanisms through suppression of cytokine expression in HSV-infected cells and through a reduction in the anti-HSV activity of interferon (IFN)-
and -
.
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Methods |
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Isolation of virus strains.
HSV-1 strain VR-3 was the parental strain used. pUC41, a plasmid for isolation of the UL41-deletion mutant, was constructed as follows: genomic DNA of VR-3 strain was digested with KpnI and EcoRI, and an 11·9 kbp fragment from UL39 to a part of the UL44 gene was cloned into pUC18 to give pUC39Kpn44Eco. A 3·9 kbp BamHI fragment and a 4·1 kbp MunI/EcoRI fragment were prepared from pUC39kpn44Eco, and cloned into the pUC18 BamHI and EcoRI sites with a BamHI/EcoRI fragment of the green fluorescent protein gene from pRSET-GFP to give pUC
41 (Fig. 1a
). pRSET-GFP was kindly supplied by Roger Y. Tsien, University of California, San Diego, CA, USA. The deletion construct, pUC
41, is deficient in the UL41 gene from 8 bp upstream of the start codon to the BamHI site in the UL41 gene. The DNA encoding the UL41 deletion mutation was excised by digestion with AatI and EcoRI and co-transfected with VR-3 DNA into Vero cells using FuGENE 6 transfection reagent (Boehringer Mannheim). The transfected Vero cells were cultured in MEM-NCS-2 containing 0·5% methyl cellulose 4000 (Nacalai Tesque) and progeny strains were isolated from plaques under a microscope. The genotype of isolated strains was verified by PCR of the genetically engineered region using primers UL41-S1 (5' GCGATATGACGTACTTAATGTAGC 3'; positions 9137791400) and UL41-S2' (5' TTAGGCCGATCCGCAGTTACAATT 3'; positions 9266992646), and the ApaI cleavage pattern of the PCR product. Of 29 plaques picked from the transfected culture, two plaques contained UL41-deficient recombinant virus. Three rounds of plaque purification yielded a pure population of the VR
41 strain.
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Southern blot analysis.
Genomic DNA extracted from 5x106 HSV-1-infected Vero cells was digested with HindIII and NotI, and used for Southern blot analysis. The GFP gene, prepared from the pRSET-GFP plasmid by digestion with BamHI and EcoRI, was used as a probe. Probes for the UL41 gene were prepared by PCR. The PCR amplification was carried out using primers UL41-NS (5' ttttcTaGACCTGACATGGGTTTGTTCG 3'; capital letters represent viral sequence positions 9264692625) and UL41-E (5' CAAGCGCGgTCGaCTGACGTTTGGG 3'; positions 9114391167). Two fragments, of 1035 bp and 471 bp, were purified from the BamHI-cleaved PCR product which had been electrophoresed on an agarose gel, and were used as probes for the 5'-end and 3'-end regions of the UL41 gene, respectively. Each DNA fragment was radiolabelled by the end-labelling method using polynucleotide kinase and [-32P]ATP. Hybridizations were performed at 65 °C overnight in 5x SSC (1xSSC: 150 mM NaCl, 15 mM sodium citrate), 1% SDS and 1x Denhardts solution. Following hybridization, the membrane was washed with 0·2xSSC in 0·1% SDS at 65 °C. The membrane was stripped with 0·2 M NaOH and neutralized with 0·2 M TrisHCl (pH 7·2) in 0·1% SDS prior to rehybridization with other probes.
Preparation of polyclonal antisera.
The UL41 coding sequence was amplified by PCR from genomic DNA of the HSV-2 186 strain using UL41f (5' attgcggccgcATGGGTCTGTTTGGCATGATGAAG 3'; capital letters represent viral sequence positions 9327593252) and UL41r (attgcggccgcCTACTCGTCCCAGAATTTAGCCAG; positions 9179791820) as the primers (Dolan et al., 1998 ). PCR products were digested with NotI and cloned into the NotI site of pET-28a (Novagen) to give pET-UL41. The plasmid, pET-UL41, was transformed into E. coli strain BL21(DE3), which following induction with IPTG, expressed 6x His-UL41 fusion protein. The UL41 fusion protein was purified using the Prep Cell system (Bio-Rad), and the purified fractions were used to immunize three rabbits, as described previously (Daikoku et al., 1997
).
Western blot analysis.
Western blotting was performed as described previously (Oshima et al., 1998 ). Briefly, proteins were electrophoretically transferred from an SDSpolyacrylamide gel to PVDF transfer membranes. Respective bound primary antibodies were detected using horseradish peroxidase-linked sheep anti-rabbit immunoglobulin G and ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
vhs Assay.
Vero cells in 35 mm Petri dishes were infected with HSV-1 strains at an m.o.i. of 10 p.f.u. per cell, and cultivated in MEM-NCS-2 supplemented with 2 µg/ml of actinomycin D (MEM-Act D). After 4·5 h of incubation in a CO2 incubator, the cultures were re-fed with MEM-Act D containing 50 µCi/ml of Pro-mix (mixture of L-[35S]methionine and L-[35S]cysteine; Amersham) and pulse-labelled for 30 min. The cells were lysed by incubation with 500 µl of SDS sample buffer (65 mM TrisHCl, pH 6·8, 2% SDS, 5% 2-mercaptoethanol) containing 100 unit/ml Benzone nuclease (Merck) at 37 °C for 30 min.
Labelling was quantified as follows: labelled proteins were precipitated by addition of trichloroacetic acid (TCA) at a final concentration of 10% (w/v) and incubation for 30 min on ice. After centrifugation for 10 min at 12000 r.p.m. using a micro-centrifuge, the supernatant was discarded and the pellet was washed three times with 10% ice-cold TCA. The incorporated 35S-radioactivity was measured in a liquid scintillation counter.
Labelled proteins were analysed by electrophoresis on a 10% SDSpolyacrylamide gel and fluorography using EN3HANCE (NEN Life Science).
Mouse model for HSV-1 infection.
The virulence of HSV-1 strains VR-3, VR41, VR
41R and the thymidine kinase (TK)-negative strain VRTK- were analysed in a mouse model for HSV-1 encephalitis as described previously (Suzutani et al., 1988
, 1995
). Groups of 10 adult (4-week-old) or newborn (3-day-old) female BALB/c mice were inoculated in the right cerebral hemisphere with 3 µl aliquots of serial 10-fold dilutions of the HSV-1 strains, and the survival rate was assessed daily for 28 days. The 50% lethal doses (LD50) for the virus strains were determined graphically.
For suppression of mouse defence functions, -rays from 137Cs were used to irradiate mice using Gammacell 40 (Atomic Energy of Canada Ltd).
The virus titre in the brains of HSV-1-infected mice was determined as follows. Mice were decapitated under ether anaesthesia, and the cerebra were removed and homogenized in 2 ml of MEM with a homogenizer. The extracts were centrifuged at 3000 r.p.m. for 10 min at 4 °C and the virus in the supernatants was titrated on Vero cells.
Quantification of mRNA.
The expression patterns of genes were semi-quantitatively monitored using Atlas human cDNA expression array I (Clontec Laboratories). The virus stocks of each HSV-1 strain used in these experiments were purified by sucrose gradient centrifugation in order to remove cellular factors. Total RNA was extracted from 5x1061x107 mock- or HSV-1-infected HEL or differentiated U937 cells at 1 h post-infection using TRIZOL reagent (Gibco BRL). mRNA was purified from the total RNA samples by oligo(dT) cellulose spin columns (5 Prime3 Prime Inc.). 32P-labelled cDNA was synthesized and hybridized to a membrane onto which 588 human DNA had been immobilized, in accordance with the manufacturers instructions. After washing, the membranes were exposed to an Imaging Plate for 2 h. Gene expression was quantified by analysing the radiogram using a Bio-Imaging Analyser BAS2000 (Fuji Photo Film).
ELISA.
The quantities of interleukin (IL)-1, IL-8 and macrophage inflammatory protein (MIP)-1
in the medium of mock- and HSV-1-infected human cell cultures (HEL, NB69 and primary macrophages) at 24 h post-infection were measured using ELISA kits (IL-1
and IL-8 immunoassay kits, BioSource International; MIP-1
immunoassay kit, R&D Systems).
Effect of vhs function on susceptibility of HSV-1 to IFN.
Susceptibility of HSV-1 strains to IFN was evaluated by a plaque reduction assay on HEL cells as previously described (Suzutani et al., 1988 ). Briefly, confluent HEL cells in 24-well plastic plates were re-fed with MEM-NCS-10 containing various concentration of IFN and cultured for 16 h in a CO2 incubator. The IFN-treated HEL cells were infected with 1020 p.f.u. of HSV-1 strains and cultured with MEM-NCS-2 supplemented with 0·5% methyl cellulose 4000 and IFN for 3 days. IFN-
and -
used in this study were natural type and kindly supplied by Sumitomo Pharmaceuticals and Daiichi Pharmaceutical Co., respectively. Recombinant IFN-
was provided by Shionogi & Co.
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Results |
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Genotypes of the isolated virus strains were analysed by Southern blot hybridization (Fig. 1a, b
). A 3·7 kbp fragment of the VR-3 strain and VR
41R strain, and a 3·6 kbp fragment of the VR
41 strain in HindIII/NotI-cleaved viral DNA were detected using a probe of the 3' end segment from the BamHI site of the UL41 gene. The identical 3·6 kbp DNA fragment was detected using a probe of the GFP gene in the VR
41 genome, while a probe from the 5' end segment of the UL41 gene, between the MunI and BamHI sites, hybridized to 3·7 kbp fragments from the VR-3- and VR
41R genomes but not the VR
41 genome. These results indicate that the genotypes of the VR
41 and VR
41R strains are those of a UL41-deficient virus and a UL41 gene rescued virus, respectively.
The phenotypes of strains were verified with Western blotting and a vhs assay (Fig. 1ce
). Western blotting using polyclonal antisera against HSV-2 UL41 polypeptide detected the 58 kDa polypeptide expressed from the UL41 gene in VR-3- and VR
41R-infected cells at 12 h post-infection but not in VR
41-infected cells (Fig. 1c
). The VR-3 and VR
41R strains reduced uniformly the synthesis of all host proteins of infected cells to 15% of the level of mock-infected cells (Fig. 1d
, e
). In contrast, no vhs activity was observed in VR
41-infected cells. These results verified by both genotypic and phenotypic characteristics that strain VR
41 is a UL41-deficient strain and that VR
41R is a revertant of VR
41 in the UL41 gene.
Virulence of HSV-1 strains in mice
The effect of vhs function on virulence was analysed by using a BALB/c mouse encephalitis model. The VR41 strain showed low virulence, depending on the age of the host mouse (Table 1
). The results suggested two possibilities for the cause of the low virulence of the VR
41 strain: (i) the VR
41 strain can replicate in dividing neurons of suckling mice but not in stationary neurons of adult mice, like the TK-deficient HSV described previously (Suzutani et al., 1995
), or (ii) the maturity of cells responsible for non-specific defences against virus replication influences the sensitivity of animals to VR
41 infection (Johnson, 1964
; Hayashi et al., 1980
). In order to clarify the cause of the age-dependent low virulence of VR
41, the effect of
-ray irradiation of mice on the virulence was examined. The LD50 of the VR
41 strain was decreased in proportion to the dose of
-ray irradiation in the host mice (Fig. 2
). At 4 Gy of irradiation, the LD50 value of VR
41 was 1·0 p.f.u. per mouse, which was the same as the LD50 values of VR-3 and VR
41R in non-irradiated mice. Irradiation with 4 Gy just on the head had little effect on the LD50 value of VR
41. On the other hand, the virulence of the TK-deficient strain (VRTK-) isolated from the same parental strain was not enhanced by
-ray irradiation, because TK-deficient HSV cannot replicate in stationary neurons. These results indicate that the major cause of low virulence is the inhibition of VR
41 replication by a
-ray-sensitive cell population(s).
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Identification and quantification of cytokine gene expression in HSV-infected human cells
To identify the cytokines secreted from VR41-infected cells, gene expression in mock-, VR-3-, VR
41- and VR
41R-infected human embryo fibroblast cells (HEL) and macrophage-like cell line U937 was screened semi-quantitatively, using Atlas cDNA expression arrays (Clontech). The amounts of mRNAs from the IL-1
, -1
, -2 to -13, -15, -17 and -18, IFN-
, -
and -
, tumour necrosis factor-
and 17 other kinds of chemokine genes could be evaluated by Atlas. Only three out of the 37 cytokine genes, the IL-1
, IL-8 and MIP-1
genes, were expressed in VR
41-infected U937 cells at 1 h post-infection (Table 2
). Expression of IL-1
and IL-8 genes was enhanced about 2-fold by VR
41-infection as compared with mock-infected cells. On the other hand, only very low levels of the mRNAs detected in VR
41-infected cells were detected in VR-3-infected cells. In HEL cells, none of the cytokines examined in Atlas were detected in HSV-infected or mock-infected cells.
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Discussion |
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The results and suggestions obtained from experiments using the mouse encephalitis model appear to be supported by evidence that enhanced expression of three cytokine genes was detected in VR41-infected human macrophage cells by screening using Atlas cDNA expression arrays (Table 2
). All of these cytokines are concerned with activation of the non-specific defence mechanism and induction of inflammation: IL-1
is an important inflammatory cytokine which activates natural killer cell activity and production of other cytokines such as IL-6 and chemokines, while IL-8 and MIP-1
, which are C-X-C and C-C chemokines, respectively, induce activation and migration of neutrophils and monocytes (Harada et al., 1994
; Baggiolini et al., 1995
). Moreover, the expression of these cytokine genes led to production of the cytokine peptides (Table 3
). It is possible to conclude from these results that one important function of vhs is evasion of host non-specific defence mechanisms by suppressing activation and migration of granulocytes and monocytes through shutting off cytokine production in HSV-1-infected cells.
The rodent brain contains two populations of macrophages, the microglia, which reside anywhere in the central nervous system (CNS), and the CNS-associated macrophages, which reside within the vascular basement membrane in close proximity to blood vessels (Stoll & Jander, 1999 ). Similar to macrophages in peripheral blood or tissues other than CNS, activated microglia and macrophages in CNS can synthesize a variety of soluble factors, including cytokines. Considering the function of microglia and CNS-associated macrophages with our result, since cytokines were produced by HSV-infected macrophages from peripheral blood but not in HSV-infected neuroblastoma cells (Table 3
), the source of cytokines produced in the HSV-infected brain would be microglia and/or macrophages activated by HSV infection. Our results support this supposition in that irradiation with 4 Gy limited to the head had little effect on the LD50 value of VR
41, because microglia and macrophages are highly tolerant to
-ray irradiation (Fabrikant, 1972
).
In the screening of gene expression in HSV-infected cells by Atlas cDNA expression arrays, we could only detect IL-8 gene expression in U937 cells with VR-3 infection (Table 2), although not only IL-8 peptide from macrophages but also IL-1
and MIP-1
peptides from macrophages, and IL-8 peptide from HEL cells were detected. These results indicated two possibilities. (i) mRNAs, which could not be detected 1 h post-infection in our assay, are expressed at a later period in HSV-infected cells. (ii) The sensitivity of mRNA detection by Atlas cDNA expression arrays may be lower than that of ELISA in the detection of peptides. In our preliminary study, we could observe 13·0 and 40·8 copies per cell of IL-8 mRNA in VR-3-infected and VR
41-infected HEL cells, respectively, after a 1 h adsorption period, by quantitative RTPCR, although these were not detected by Atlas. This expression of the IL-8 gene was observed only at the very early stage of infection (data not shown) and part of the down-regulation at a later stage of infection would be caused by the delayed shutoff function of HSV. These observations seem to support the latter possibility described above. Therefore, it is possible that VR
41-infection enhanced the production of a number of cytokines in macrophages of which we identified only IL-1
, IL-8 and MIP-1
.
Recently, a number of studies have been performed on the molecular mechanisms of virus evasion of host defence systems. The evasive functions of four proteins encoded by the HSV genome have previously been demonstrated, i.e. a complex of glycoprotein E (gE; encoded by the US8 gene) and gI (encoded by the US7 gene) forms an Fc receptor on the surface of infected cells and virions, and the Fc receptor forms antibody bipolar bridges resulting in the escape from neutralization of virions and infected cells by complement and antibody-dependent killer cells (Para et al., 1982 ; Johansson et al., 1986
; Johnson & Feenstra, 1987
; Frank & Friedman, 1989
). Also, complement activity is suppressed by gC (encoded by UL44), which is a C3b receptor (Friedman et al., 1984
; McNearney et al., 1987
; Van Strijp et al., 1988
). ICP47, one of the immediate-early proteins encoded by the US12 gene, inhibits antigen presentation on the surface of infected cells by inhibition of peptide transporter (TAP), mediating the escape from cytotoxic T cells (York et al., 1994
; Früh et al., 1995
; Ahn et al., 1996
; Tomazin et al., 1996
). In this study, we showed that the vhs protein is a fifth protein mediating evasion of host defence mechanisms.
It is assumed that the vhs function affects all mRNAs, including viral mRNAs; therefore, vhs might be able to target modifications of various cellular functions and viral functions. In this study, we focused on the early stage of primary HSV infection and showed one of the vhs functions to be evasion of non-specific defence mechanisms. However, this evasion might be only a part of the vhs function even in evasion from host defence, since a previous report showed that vhs reduces major histocompatibility complex (MHC) class I molecules on the plasma membrane of HSV-infected cells and a vhs-deficient strain induces strong immunity as a vaccine strain (Hill et al., 1994 ; Walker & Leib, 1998
; Walker et al., 1998
). A radical strategy like that of vhs, which non-specifically suppresses the synthesis of various proteins, regardless of whether they are beneficial or harmful factors, would be possible in the replication cycle of HSV, which is completed in 12 h, but not in cytomegalovirus replication. Further studies of the roles of vhs will advance our understanding of virushost interactions.
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Acknowledgments |
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Received 3 November 1999;
accepted 8 March 2000.