1 Istituto di Genetica Molecolare, CNR, via Abbiategrasso 207, I-27100 Pavia, Italy
2 Servizio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy
3 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
4 Max von Pettenkofer-Institut, Lehrstuhl für Virologie, LMU-München, München, Germany
5 Department of Medicine and Surgery, San Paolo Hospital, University of Milano, via A. di Rudin 8, I-20142 Milano, Italy
Correspondence
Andrea Gallina
at Istituto di Genetica Molecolare
gallina{at}igbe.pv.cnr.it
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ABSTRACT |
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Present address: Laboratorio di Terapia Genica e Molecolare, IFCCNR, Area della Ricerca, Via G. Moruzzi, 56124 Pisa, Italy.
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INTRODUCTION |
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The class I ribonucleoside diphosphate (ribonucleotide) reductases (RR), which catalyse a limiting step in de novo deoxyribonucleotide (dNTP) synthesis, are essential for DNA replication of both eukaryotic cells and the DNA viruses that infect them (Jordan & Reichard, 1998; Stubbe et al., 2001
). RR holoenzymes comprise a catalytic large (R1) subunit, and a small (R2) subunit required for enzyme activation (Jordan & Reichard, 1998
). Like many large DNA viruses, the
- and
-herpesviruses encode both subunits of a viral RR, which is important for virus replication in resting or post-mitotic cells (Jacobson et al., 1989
; Idowu et al., 1992
; Heineman & Cohen, 1994
; Aurelian, 1998
) because these have minute dNTP pools and virtually no endogenous RR (Engstrom et al., 1985
; Jordan & Reichard, 1998
; Chabes & Thelander, 2000
). In addition, the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) R1 proteins bear an N-terminal extension (see supplementary data Fig. 1
at JGV Online, http://vir.sgmjournals.org), which has been suggested to contain an intrinsic protein kinase (PK) activity (Chung et al., 1989
; Paradis et al., 1991
; Cooper et al., 1995
), although this view has been challenged (Langelier et al., 1998
; Conner, 1999
). HSV-2 R1 (ICP10) enhances the function of HSV-2 immediate-early (IE) proteins, plays a role in HSV-2 latency reactivation (Smith et al., 1998
) and protects infected cells from chemically induced apoptosis (Perkins et al., 2002
; Langelier et al., 2002
), in correlation with MEK/MAPK pathway activation (Smith et al., 2000
).
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The role of HCMV R1 (UL45) has been recently assessed in the infection context. The UL45-KO derivative of an endotheliotropic strain grew in human umbilical vein endothelial cells with normal kinetics and no sign of apoptosis (Hahn et al., 2002). Here, we characterize UL45 expression kinetics in cells infected with the non-endotheliotropic strain AD169, and define UL45 as a late, virion-associated protein. A UL45-KO mutant of the same strain is defective in viral particle accumulation at low multiplicities of infection (m.o.i.), and in spreading in fibroblasts. This is unrelated to dNTP production, as both mutant and parental virus induce cellular RR subunits and accumulate dNTPs with similar efficiency. The protection from Fas-induced apoptosis is minimally reduced in UL45-KO-infected fibroblasts relative to the parental virus-infected cells. UL45 and M45 functions are not identical, a difference which may be a result of their divergent N-terminal sequences.
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METHODS |
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Detergent and protease treatment of purified virions.
Purified viral particles of HCMV strains AD169 and RV d65 (an AD169 derivative carrying a disrupted UL83; Schmolke et al., 1995) were obtained previously, and tested free from infected cell debris by Western blot analysis with anti-UL44 and anti-
-tubulin antibodies (Gallina et al., 1999
). Particles dissolved in 10 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·5 % Triton X-100 were incubated on ice for 30 min. The detergent-insoluble fraction was separated by centrifugation at 100 000 g for 1 h, at 4 °C. Alternatively, virions suspended in the same buffer, with/without added Triton X-100, were treated with 50 µg trypsin ml-1 at 30 °C for 15 min. The reaction was stopped by addition of a protease inhibitor cocktail (Complete, Roche) and incubation on ice for 15 min. Ten µg of virion protein per treatment was analysed by immunoblot.
Nucleic acids purification and analysis.
Total DNA from HELF was purified with the QIAamp DNA mini kit (Qiagen). For Southern blotting, EcoRI-digested DNA was electrophoresed on a 0·6 % agarose gel and blotted onto Hybond-N+ (Amersham Pharmacia). The membrane was hybridized with an [32P]dCTP-labelled UL45 probe and exposed to a phosphorimager screen.
For viral DNA quantifications, fivefold dilutions of purified DNA (extensively treated at 37 °C for 1 h with 40 µg RNaseA ml-1 to remove contaminating RNA) were immobilized onto Hybond-N+ membrane. This was hybridized with a 1·5 kbp HCMV-specific probe covering UL83, and the signal quantified by phosphorimaging. The stripped membrane was rehybridized with a probe for human GAPDH to normalize data.
For transcription analysis, RNA from infected/mock-infected HELF was subjected to two rounds of purification on RNeasy columns (Qiagen), interposing a treatment with 1 U RQ DNase (Promega) (µg RNA)-1 at 30 °C for 15 min in the supplied buffer. RNA (20 ng) was amplified with primer pairs appropriate for UL45 (primers 2 and 3; these and the other primers mentioned hereafter are listed in Table 1), R1, R2 (Pavloff et al., 1992
), p53R2 (Tanaka et al., 2000
) and other viral or cellular transcripts, with/without previous reverse transcription, according to the Access-RT-PCR kit (Promega) protocol.
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Plasmid construction and recombinant protein expression.
PCR products were generated with Pfu polymerase and sequenced after cloning to rule out unwanted mutations. Plasmid pGex-NTD was obtained by cloning into pGex4T2 (Amersham Pharmacia) UL45 codons 2278, amplified with primers 2 and 3 from AD169 DNA. IPTG-induced E. coli DH5 cells harbouring pGEX-NTD were processed for inclusion body purification (Sambrook et al., 1989
). Washed GST-UL45(2278) inclusion bodies were suspended in PBS and used for chicken immunizations.
Full-length UL45 coding sequence (primers 14) was inserted into a pcDNA4/HisMAX-A (Invitrogen) derivative, which lacks an NheIBamHI fragment encoding a polyhistidine tract. The final construct was transfected into HEK-293 cells (2 µg DNA per 1x106 cells) according to the calcium phosphate protocol.
The same UL45 insert was cloned into the retroviral vector pLEGFP-C1 (Clontech) in place of the Neo cassette to create retro-UL45. Retro-UL45 or the void vector (retro-cont) were transfected into 293gp/bsr packaging cells together with plasmid pVG, which expresses vesicular stomatitis virus G protein for envelope pseudotyping (Somia et al., 2000). Retrovirus in transfection supernatants was titrated by infecting 293-HEK monolayers and visualizing enhanced green fluorescence protein (EGFP)-positive cells under an epifluorescence microscope.
Immunoblots and radioimmunoprecipitations.
Proteins in infected/mock-infected HELF extracts were quantified by a colorimetric method (BCA, Pierce). Balanced amounts of total protein were analysed by Western blot and radioactive immunoprecipitation (Sambrook et al., 1989), performed according to standard procedures (for details and the list of antibodies employed, see supplementary data on materials at JGV Online, http://vir.sgmjournals.org).
Indirect immunofluorescence.
HELF seeded on coverslips (1x105 cells cm-2) were infected, mock-infected or treated with 0·2 µg doxorubicin ml-1 for 48 h to induce DNA damage and p53R2 nuclear localization. Monolayers were fixed in 3·8 % (w/v) paraformaldehyde in PBS at 4 °C for 30 min. After a 10 min poration in 0·1 % (v/v) Triton X-100 in PBS, cells were incubated for 20 min at 37 °C in a humid chamber with 6 µg anti-p53R2 ml-1 and 2 µg anti-pp72/pp86 ml-1 in 0·1 % (v/v) Triton X-100/10 % (v/v) FBS in PBS. Cells were washed and incubated with secondary antibodies (fluorescein isothiocyanate-conjugated anti-mouse IgG and tetramethylrhodamine-conjugated anti-rabbit IgG; Chemicon), 200 ng 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) ml-1 in the same buffer. After a final wash cells were mounted in Prolong (Molecular Probes).
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RESULTS |
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These data assign UL45 to the HCMV -1 genes, whose mRNA translation, rather than transcription, is postponed to the onset of genome replication (Mocarski, 1996
). The late accumulation kinetics encouraged us to investigate whether pUL45 becomes incorporated into HCMV particles. Significant amounts of pUL45 were indeed found in gradient-purified HCMV particles (Fig. 1C
). The UL45 protein remained associated with particles stripped off the envelope by exposure to a non-ionic detergent, but this treatment rendered it completely sensitive to trypsin. A pUL45 band was similarly detected in virions of the RVd65 mutant, an AD169 strain derivative (Schmolke et al., 1995
) that lacks a functional UL83 open reading frame and that no longer produces the UL83 protein (pp65)-rich dense bodies. Taking sedimentation with detergent-insoluble components as proof of association with capsid-tegument, protease sensitization in the presence of detergent as proof of association with tegument, and presence in dense body-free particles as proof of incorporation into infectious virions, we infer that, akin to HSV-2 ICP10 (Smith & Aurelian, 1997
), pUL45 is exported from the cell as a virion tegument component. As immunofluorescent analysis of infected cells showed that pUL45 is cytoplasmic (data not shown), pUL45 joins the list of tegument proteins, the extra nuclear localization of which proves a re-envelopment step in HCMV virogenesis (Sanchez et al., 2000
a, b).
The UL45 null mutants are viable but exhibit a replication deficit at a low m.o.i.
Recombinant viruses carrying the UL45 orf interrupted by a transposon (Tn) insertion were isolated by screening a Tn library of the cloned AD169 genome (Borst et al., 1999; Hobom et al., 2000
; for details see supplementary data on materials at JGV Online, http://vir.sgmjournals.org). Two Tn insertion mutants were identified (K21E4 and K19H1). Direct Bac sequencing mapped Tn insertions at nucleotide 59180 (K21E4) and 58642 (K19H1) (see supplementary data Fig. 3A
C at JGV Online, http://vir.sgmjournals.org). Tn insertions truncate UL45 at codon 64 and 244, respectively (see supplementary data Fig. 1
at JGV Online). Reconstituted virus (RV) was recovered by transfection of Bac DNA into fibroblasts. Comparison of RV-AD169 (wild-type), RV-K19H1 and RV-K21E4 by Southern blot (see supplementary data Fig. 3D
at JGV Online) and PCR-sequencing (data not shown), confirmed a stable Tn insertion in the virus mutants. We conclude, in agreement with earlier work (Hahn et al., 2002
), that the UL45-encoded protein is dispensable for replication of HCMV in primary HELF. Subsequent work focused on RV-K21E4, in which UL45 truncation after a few codons minimizes the risk of a hypomorph variant.
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Single step (high m.o.i.) HELF infections showed overlapping growth curves for parental and mutant viruses (Fig. 2B), as reported for the UL45-KO mutant of the endotheliotropic HCMV bacvirus (Hahn et al., 2002
). Nonetheless, an appreciable reduction of mutant virus accumulation was observed under low-m.o.i. (0·1 IU per cell) conditions. RV-AD169 and RV-K21E4 growth curves diverged at 5 days p.i. and the final titre of the mutant (both cell-associated and secreted) was >1 log lower (Fig. 2C
). To test whether the missing UL45 product was the cause of the growth difference, a retrovirus vector was used to trans-complement UL45 in HELF. The recombinant retrovirus, here referred to as retro-UL45, expresses the UL45 coding region from the LTR. Additionally, it expresses the EGFP protein from an internal promoter. UL45 protein synthesis following transduction with 5 retrovirus units per cell was monitored by Western blot. UL45 protein was first detected at 24 h p.i.; it accumulated to a plateau level at 48 h p.i. (Fig. 3
A) and was stably expressed for at least three subsequent doublings of transduced cells (not shown). EGFP was equally expressed by cells infected with retro-UL45 and with void retroviral vector (retro-cont). EGFP-positive HELF were isolated by fluorescence-activated cell sorting and used for HCMV infections. RV-AD169 and RV-K21E4 growth curves were reassessed in retro-UL45 and retro-cont transduced cells. While RV-AD169 growth was the same in the two cell hosts, RV-K21E4 replicated more efficiently in retro-UL45 transduced cells, with final titres approaching those of wild-type (Fig. 3B
). Thus, the growth defect of UL45 mutant can be ascribed to the disruption of UL45.
The ability of the mutant to form plaques was also investigated. Cells were infected at a low m.o.i., with anti-HCMV antibodies (pooled human sera) added to the medium to prevent secondary infections. Although average plaque extension was similar, the mutant failed to form large plaques (all plaques in the 215 cells per plaque interval at 9 days p.i.), contrary to the parental (>20 % of the plaques in the 1630 cells per plaque interval at 9 days p.i.; data not shown).
UL45 knockout leaves dNTP synthesis unaltered
To test whether the absence of pUL45 was directly hindering HCMV DNA replication due to a dNTP shortage, the kinetics of dNTP accumulation were examined in RV-AD169 and RV-K21E4-infected HELF (Fig. 4A). Infection resulted in the rise of the dATP pool, and a very large expansion of the dTTP pool, in agreement with a previous report (Biron et al., 1986
). A peak was detected at 2 days p.i., possibly in relation to a cellular RR peak (see below). However, the dNTP pool characteristics remained very similar in RV-AD169 and RV-K21E4-infected cells, throughout the entire course of infection.
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HCMV rescues the three cellular RR subunits
The above results indicate that dNTP synthesis and HCMV DNA replication are not primarily affected by UL45 knock out, implying that cellular RR is responsible for the provision of dNTPs to replicating viral DNA. Previous work (Chabes & Thelander, 2000; Engstrom et al., 1985
; Jordan & Reichard, 1998
) has shown that mammalian RR is activated during the cell cycle. The R2 subunit acts as the limiting factor, restricting RR activity to the SG2 phase. By contrast, both R1 and R2 proteins are virtually undetectable in resting cells. Thus, HCMV infection must be able to force the expression of both subunits of human RR, in non-replicating cells. The impact on cellular RR mRNAs was investigated in HCMV infected resting HELF at 372 h p.i. By 3 h p.i., the virus had induced a marked host R2 transcription that continued through later times (Fig. 5
A), whereas R1 transcripts were already present in non-infected cells. At the protein level (Fig. 5B
), both cellular subunits were induced with similar kinetics, from almost undetectable levels to a peak at 48 h p.i., followed by a decrease at later times. A similar pattern was observed in RV-K21E4-infected cells. Cellular R1/R2 synthesis is then induced early following infection, with a post-transcriptional component for R1, and independent of UL45 expression.
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A difference at the limit of statistical significance did emerge only when apoptosis was provoked by exposing the infected HELF to anti-Fas antibody plus CHX. Cell survival amounted to approximately 50 % of the RV-AD169 infected monolayer and 26 % of RV-K21E4 infected cells (Fig. 7). To confirm that the viral protein was responsible for the parental virus advantage, and to test whether UL45 could trigger the antiapoptotic effect in the absence of infection, the experiment was repeated in retro-UL45-transduced cells. In this setting, survival of parental- and mutant-infected HELF was indistinguishable, but pUL45 was unable to protect uninfected cells (Fig. 7
). Thus, pUL45 confers a marginal but specific protection against death receptor signalling in the context of virus infection, but does not display the properties of a dominant antiapoptotic factor.
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DISCUSSION |
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To offset the loss of a virus-encoded RR, HCMV effectively rescues cellular RR activity in resting HELF, by upregulating all known cellular RR subunits, according to our data. In the case of SG2 phase RR subunits, our data extend the evidence from a DNA microarray study to the infection context, showing that both R1 and R2 genes are induced, together with many other S-phase genes under E2F1 transcriptional control, in human fibroblasts expressing HCMV IE2-pp86 (Song & Stinski, 2002). While R1R2 induction fits into the general picture of cell cycle subversion operated by HCMV IE proteins, conducive to the expression of S phase replication proteins (reviewed by Fortunato et al., 2000
; Femington, 2001
; Kalejta & Shenk, 2002
), our finding that p53R2 is also induced and targeted to the nucleus of infected HELF is intriguing because p53R2 is related to DNA repair, not replication; the p53R2 gene is indeed the first p53 transcriptional target directly linking p53 to repair (Tanaka et al., 2000
; Yamaguchi et al., 2001
). In HCMV infected fibroblasts p53 protein is stabilized, but its ability to activate transcription is blocked (Kalejta & Shenk, 2002
). Our data do not contradict this point, as p53R2 transcript levels do not increase during infection. Nevertheless, HCMV is capable of circumventing p53 blockade by post-transcriptionally inducing p53R2 whether this is by enhancing mRNA translation or by stabilizing the protein remains to be established. A related question is whether p53R2 nuclear targeting is a consequence of the activation of a DNA damage signal in the infected cell, or is obtained ad hoc by virus encoded factor(s). In either case, the concentration of an RR activating subunit in the proximity of HCMV replication factories may help to boost the supply of precursors to the viral DNA synthesis and repair machinery.
Three phenotypes could be associated with the UL45-KO mutant RV-K21E4. A multi-step growth analysis highlighted a 1 log (50-fold) growth deficit. To formally assign the growth defect to UL45 inactivation, the viral protein was complemented in trans via a retrovirus vector. The trans-complemented mutant grew like the parental, showing that the phenotype can be safely ascribed to pUL45 absence and not to polar effects on the surrounding genes, UL44 and UL46 (minor capsid protein), both of which are essential for virus replication (Mocarski, 1996; Pari & Anders, 1993
; Pari et al., 1993
; Ripalti et al., 1995
). This also rules out adventitious mutations introduced during bacvirus manipulations in the prokaryotic host. A possibly related feature is that UL45-KO mutant forms plaques less efficiently than the parent. As both phenotypes are typical of defects in virus egress and/or cell-to-cell spread, pUL45 might be involved in some aspect of virion assembly and/or penetration. In agreement with this hypothesis, pUL45 is a late phase product and is present in purified virions as a tegument component, with the potential to enhance virion infectivity or to act as a factor delivered to the host cell upon infection.
An additional feature of the mutant is that it confers on the infected cells a slightly (<2-fold) reduced protection from anti-Fas-induced apoptosis relative to the parental. This difference was not observed with agents acting via the mitochondrial pathway (etoposide and menadione; data not shown). Also, non-infected fibroblasts synthesizing pUL45 were not protected from Fas activation. Four HCMV products have been identified that counter experimentally induced apoptosis: IE1-IE2 proteins, which interfere with p53 function and activate the phosphatidylinositide 3'-OH kinase-Akt pathway (Zhu et al., 1995; Yu & Alwine, 2002
); vMIA (UL37x1), which acts at the mitochondrial level (Goldmacher et al., 1999
); and the caspase-8 inhibitor vICA (UL36) (Skaletskaya et al., 2001
). vICA is inactivated by a mutation in the HCMV strain AD169varATCC, which explains the incomplete protection imparted by the parental virus in our study (Skaletskaya et al., 2001
; this study). All these proteins confer a substantial protection when assayed independent of virus infection; that is, they are strictly speaking antiapoptotic. By contrast, the effect of pUL45 is weak, selective for Fas-induced apoptosis among the tested stimuli, and restricted to infected fibroblasts. Perhaps, this mild pro-survival activity constitutes a side-effect of a physiological role. MAPK/ERK and p38 MAPK are activated by HCMV (Rodems & Spector 1998
; Johnson et al., 2000
, 2001
), and the impact of pUL45 on this and other transduction pathways warrants exploration.
A relevant question is to what extent does pUL45 differ from M45. A reduction in progeny virus titre similar to that described here is apparent in MCMV M45-KO mutants grown in murine fibroblasts (Brune et al., 2001). Thus, the loss of RR activity and a measurable impact on virus propagation in fibroblasts are seemingly general traits of
-herpesvirus R1 proteins. The report by Lembo et al. (2000)
that dNTP pools in MCMV-infected fibroblasts resist the shift induced by exogenous thymidine has been taken as an indication that MCMV synthesizes an active, allosterically unregulated, R1. However, the effect might involve other levels of dNTP homeostasis, e.g. nucleotide excretion.
On the other hand, M45 has been recognized as a factor preventing cell-autonomous apoptosis in MCMV-infected endothelial cells (Brune et al., 2001), while its antiapoptotic potential when expressed alone has not been analysed. Growth in endothelial cells could not be checked for the mutants described here (AD169 strain is non-endotheliotropic), but the study of an UL45-KO mutant of an endotheliotropic strain failed to show apoptosis of infected human umbilical vein endothelial cells (Hahn et al., 2002
). In
-herpesvirus R1 proteins, the RR region of homology (a central all-alpha and distal barrel domain; see supplementary data Fig. 1
at JGV Online, http://vir.sgmjournals.org), common to the entire R1 family, is preceded by a unique N-terminal region subdivided into an N-terminal stretch heterogeneous in both size and sequence, followed by a conserved
110 aa homology box (BRH1). The long (385 amino acids) M45 N-terminal stretch has interspersed tracts of homology to HSV-2 ICP10 that might account for the antiapoptotic function. These are not detected in the 167 amino acid pUL45 N-terminal stretch. It is thus conceivable that distinct, specialized functions reside in pUL45 and pM45 N termini, while the BRH1 and RR homology could exert conserved function(s). In transfected HEK-293 cells, full-length pUL45 is targeted to the insoluble, cytoplasmic cytoskeleton, whereas a variant devoid of the RR region is cytosolic (M. Patrone, unpublished data). UL45 RR domains might then exert a targeting function, possibly to permit incorporation into virion tegument.
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ACKNOWLEDGEMENTS |
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Received 19 June 2003;
accepted 3 September 2003.