1 Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
2 Max von Pettenkofer Institut, Abteilung Virologie, LMU-München, Germany
Correspondence
Giuseppe Gerna
g.gerna{at}smatteo.pv.it
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ABSTRACT |
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INTRODUCTION |
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More recently, our groups, based on the cloning in bacterial artificial chromosome (BAC) of the genome of a clinical HCMV isolate (FIX-BAC) and the systematic mutagenesis of the reconstituted virus referred to as RV-FIX (Hahn et al., 2002), have been able to identify in the UL131128 genes the genetic region of HCMV driving both endotheliotropism and virus transfer to leukocytes (Hahn et al., 2004
).
Here, we document that only viral variants or RV-FIX mutants with a Huv+ Leuk+ phenotype are also DC-tropic (DC+), following co-culture of DC with HUVEC infected with the relevant strains. DC-tropism is defined as the property of HCMV to productively infect DC. These data lead to the conclusion that UL131128 gene products are required not only for endotheliotropism and virus transfer to leukocytes, but also for DC-tropism. In addition, a potential immunological application of these virologic findings to the study of HCMV-specific T-cell-mediated immune response is preliminarily reported by showing the HCMV-specific CD4+ and CD8+ T-cell responses to HCMV-infected DC.
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METHODS |
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HCMV strains, deletion mutants, natural variants and revertants.
The following two groups of HCMV strains were tested for their ability to infect and replicate productively in DC. Group A consisted of Huv+ Leuk+ HCMV strains grown in HUVEC (Hahn et al., 2004) and included: (i) four recent clinical isolates adapted to growth in HUVEC (VR1814, VR3480, VR6110 and VR6952); (ii) RV-FIX and RV-FIX deletion mutants missing single genes outside the UL131128 locus (RV-FIX
UL45,
UL127,
UL132); and (iii) HCMV revertants (VR1814 rev, VR3480 rev, AD169 rev and Towne rev) which had reacquired both EC-tropism and leukocyte transmissibility after loss of both properties in HELF (Gerna et al., 2002b
, 2003b
). Group B consisted of Huv Leuk HCMV strains grown in HELF and included: (i) clinical isolates extensively propagated (Revello et al., 2001
) in HELF (VR1814 and VR3480); (ii) laboratory strains (AD169 and Towne); and (iii) RV-FIX deletion mutants missing either the entire UL131128 locus or each one of the three ORFs (Hahn et al., 2004
). In detail, RV-FIX
UL132128 (lacking the UL131128 region including a predicted polyA signal of UL132, thus knocking out UL132128), RV-FIX
UL131K, RV-FIX
UL130 and RV-FIX
UL128 (Hahn et al., 2004
) had been shown to have lost both EC-tropism and virus transfer to leukocytes (both monocytes and PMNL).
Restriction fragment length polymorphism (RFLP) analysis.
Genomic regions UL54 (DNA Pol), UL55 (gB), UL123 (IE-1) and ULb' (UL144 to UL148) of RV-FIX at passages 14, 51 and 175 on HUVEC were amplified by PCR using the primer pairs previously reported (Revello et al., 2001; Gerna et al., 2003b
) and then cleaving PCR products with two to four of the following endonucleases: HaeIII, MspI, HinP1I, AluI and BstUI (New England Biolab). RFLP patterns were compared by agarose gel electrophoresis.
DC infection by co-culture.
Immature DCs were counted and 2·0x106 cells were co-cultured overnight with HUVEC grown in a T12 flask and infected with Huv+ Leuk+ HCMV strains or mutants, at an m.o.i. of 5. Similarly, immature DCs were co-cultured with HELF infected with Huv Leuk HCMV strains or mutants. Co-cultured DCs were removed from infected cell monolayer by gentle pipetting and then washed and migrated for 4 h in a 8 µm-pore-size Transwell chamber (Costar) inserted into wells containing 107 M of the chemotactic peptide FMLP (Sigma). DCs were migrated to remove infected HUVEC or HELF detached from the cell monolayer. Migrated cells were washed five times and counted. In general, 3·0x1055·0x105 DCs suspended in RPMI-10 % FBS supplemented with GM-CSF were seeded into one well of a 24-well microplate. The supernatant from the last washing (considered the 24 h harvest) and supernatants from DC cultures collected 4 and 7 days after onset of co-culture were titrated for virus infectivity. Tenfold dilutions of each harvest were centrifuged onto HELF monolayers, grown in shell vials, for 40 min at 600 g. The following day, coverslips were stained for the HCMV major immediate-early protein p72 (Gerna et al., 1990) using a pool of mAbs reactive to different epitopes of p72 (Gerna et al., 2003a
). In addition, approximately 5·0x104 DCs were cytocentrifuged onto a glass slide and stained for pp65 and p72 at 3 and 24 h, and for p72 and gB at 4, 7 and 10 days p.i., using the relevant mAbs and the immunofluorescence technique (Gerna et al., 2003b
; Hahn et al., 2002
).
DC infection with cell-free HCMV preparations.
When DCs were infected with cell-free Huv+ Leuk+ HCMV preparations (obtained after virus adaptation to growth in HUVEC) or Huv Leuk HCMV strains (propagated in HELF), about 1·0x106 immature DCs were incubated overnight with clarified medium from infected HUVEC or HELF cultures, respectively, at an m.o.i. of 5. Cells were washed five times, counted and cultured in one well of a 24-well microplate using RPMI-10 % FBS supplemented with 800 U GM-CSF ml1. The supernatants from the last washing (harvest of day 1) and supernatants collected at 4, 7, 10, 14 and 18 days p.i. were titrated for virus infectivity, as reported above. In parallel, at each time point, 5x104 DCs were cytocentrifuged and stained as reported above.
Co-culture of infected DC and autologous non-adhering PBMC.
DCs, following 24 h incubation (unless otherwise indicated) with Huv+ Leuk+or Huv Leuk cell-free HCMV or the relevant inactivated (by heating 5 min at 56 °C) crude viral antigen preparations, were co-cultured overnight with thawed non-adhering autologous PBMC at a ratio of 1 : 20 (5·0x104 DC : 1·0x106 non-adhering PBMC). Co-culture medium was RPMI-10 % FBS supplemented with 10 µg brefeldin A ml1 (Sigma) to prevent release of cytokines. Cell cultures were incubated overnight at 37 °C in 5 % CO2 atmosphere.
Flow cytometry analysis.
DCs, following differentiation and prior to HCMV infection, were routinely stained with FITC-conjugated anti-CD14 (Immunotech) and PE-conjugated anti-CD1a (Caltag) mAbs, supplemented with a pool of human sera, for 30 min in ice. mAb anti-CD14 acted as a monocyte marker, and mAb anti-CD1a as a dendritic-cell marker. In parallel, cells were stained with the relevant isotype-matched control mAb. Surface marker expression was then analysed using a FACScalibur flow cytometer (Becton-Dickinson). DC phenotype following co-culture, migration and further incubation was also determined.
Following incubation with infected DC, PBMCs were washed and stained with FITC-conjugated anti-CD8 (Caltag) and TC-conjugated anti-CD4 (Caltag) mAbs in 100 µl PBS containing 5 % FBS and 5 % immunoglobulin for 30 min in ice. Cells were then washed in the same buffer and fixed for 15 min (FIX and PERM; Caltag). Following washings, cells were permeabilized and stained with PE-conjugated anti-interferon (IFN) gamma (IFN-) mAb (Caltag) for 45 min. IFN-
induction was utilized as the measure of the immune response in CD4+ and CD8+ T cells. Then cells were resuspended in 1 % paraformaldehyde and analysed in a FACScalibur flow cytometer. HCMV-specific CD4+ and CD8+ T cells were determined on a minimal number of 3·0x104 viable CD4+ or CD8+ T cells and their frequencies were calculated by subtracting the value of the control sample incubated with mock-infected endothelial cell culture medium (consistently
0·05 %) from the test value. To determine the total number of HCMV-specific CD4+ and CD8+ T cells, the percentages of HCMV-specific T cells positive for IFN-
were multiplied by the relevant absolute CD4+ and CD8+ T cell count, which was determined by a direct immunofluorescent flow cytometry method (Ortho Diagnostic Systems), as reported (Gerna et al., 2001
).
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RESULTS |
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Immunofluorescent images showing the expression of immediate-early and late HCMV antigens in DC infected by a recent clinical isolate (VR1814 Huv+ Leuk+), one strain long-term propagated on HELF (VR1814 Huv Leuk), RV-FIX (generated using the genetic background of VR1814), RV-FIXUL132128 and RV-FIX
UL132 are shown in Fig. 2
.
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The kinetics of the viral protein expression during virus replication is shown in Fig. 3(b) (i)(viii). The numbers of DC positive for both pp65 (i and iii) and p72 (ii and iv) slightly increased from 3 h (i and ii) to 24 h (iii and iv) p.i. At 96 h and 168 h p.i., gB was abundantly expressed in the cytoplasm of DC, while cytopathic effect was progressing (v and vi). At 10 days p.i. DC started undergoing lysis (vii). No viral protein was expressed when DCs were incubated with Huv Leuk virus strains or mutants (viii).
Huv+ Leuk+ HCMV strains generate syncytia in HUVEC at high m.o.i.
When VR1814 infected HUVEC monolayers at high m.o.i. (around 10), syncytial formations started appearing shortly after infection (13 h), reaching conspicuous number and size at 24 h p.i. (Fig. 4a). VR1814 rev behaved similarly. On the contrary, no syncytial effect was observed in HUVEC expressing the UL131128 gene region (Hahn et al., 2004
) when infected with Huv Leuk VR1814. Huv Leuk virus strains used at the same m.o.i. did not yield syncytial formations (Fig. 4b
).
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A representative example of the FACS analysis leading to quantitative determination of CD4+ and CD8+ HCMV-specific IFN- producing T cells is given in Fig. 5(a) and (b)
. The median frequency of HCMV-specific CD4+ and CD8+ IFN-
producing T cells in 21 HCMV-seropositive and six HCMV-seronegative healthy blood donors following 1618 h activation by autologous DC infected 24 h in advance with HUVEC-adapted cell-free VR1814 virus strain, is reported in Fig. 5(c)
. The percentage of IFN-
producing T cells showed a wide range of variability in different subjects for both CD4+ and CD8+ HCMV-specific response. However, a clear-cut separation was observed between immunocompetent HCMV-seropositive and HCMV-seronegative individuals. In fact, while the number of CD4+ and CD8+ IFN-
producing T cells was negligible (consistently <0·20 cells µl1) in non-immune subjects, it reached median levels of 5·13 (0·6735·57) and 2·90 (0·4912·36) cells µl1, respectively, in immune persons. Therefore, subjects with
0·4 µl1 blood HCMV-specific CD4+ or CD8+ T cells were considered as responders' to the HCMV stimulus for the relevant T-cell subset, whereas subjects with <0·2 µl1 blood HCMV-specific CD4+ or CD8+ T cells (mean value±2 SD of six HCMV seronegative donors) were considered as non-responders. Finally, subjects with an intermediate value were considered as equivocal responders' (Fig. 5c
, grey zone).
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DISCUSSION |
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We have recently documented that UL131128 genes represent genetic determinants of both EC-tropism and HCMV transfer to leukocytes (Hahn et al., 2004). In fact, all recent clinical HCMV isolates are provided with both biological properties, whereas all deletion mutants missing the entire locus or the single ORFs of the locus, as well as the natural variants carrying mutations in the same locus, are deprived of both properties. In addition, it was shown that tropism competence can be re-acquired in Huv Leuk natural variants following re-adaptation to growth in HUVEC and reversion of UL131128 mutations naturally acquired (Hahn et al., 2004
). Finally, loss of EC-tropism and leukocyte transfer could be complemented in HUVEC transduced with the UL131128 individual genes from Huv+ Leuk+ VR1814 and infected with the relevant Huv Leuk viral variants (Hahn et al., 2004
). However, a UL24-deletion mutant of Towne was recently found to be defective in growth in microvascular endothelial cells (Dunn et al., 2003
) with respect to the parental Towne-BAC. These data seem to suggest that other determinants of EC-tropism may exist, which are functionally only active in specific cell types. In contrast with these data, we found Towne not infectious for HUVEC unless a 2 nt insertion in UL130 mutation was reverted (Hahn et al., 2004
). This discrepancy may be due to the different specificity of different endothelial cell systems or may be associated with an unpredicted reverse mutation in UL130 of the Towne strain cloned as BAC by Dunn et al. (2003)
. However, as a general consideration, the finding that RV-FIX exhibits supernatant-associated DC-tropism only after extensive adaptation to growth in HUVEC strongly suggests that additional genetic alterations have occurred during this adaptation, which are responsible for cell-free DC-tropism of HCMV strains.
In this report, we demonstrate that all HCMV strains or natural variants or deletion mutants tropism competent for HUVEC and leukocytes are also able to productively infect DC. Since release of viable virus from infected HUVEC occurs only after 2030 passages, infection of DC by virus strains or variants at earlier passages can only take place by co-culture of DC with infected HUVEC. Infection of a high proportion of DC by cell-free virus released from infected HUVEC can only be achieved with an m.o.i. of 110, as in the case of the VR1814 and RV-FIX of this study.
Since the DC used in this study are monocyte-derived and it has been previously shown that monocytes (Waldman et al., 1995) as well as PMNL (Gerna et al., 2000
) and HUVEC interact closely and bidirectionally by transferring and receiving viral and cellular material, it is not surprising that DC somewhat behave as leukocytes of the myeloid lineage with respect to endothelial cells. Three steps are needed for completion of this interaction: attraction, adhesion and fusion. Attraction is mediated by cellular and viral chemokines. In this respect, the UL128 gene product is indicated as a potential CC chemokine (Akter et al., 2003
). Then adhesion could occur through interaction between Mac-1 (CD11b/CD18) and ICAM-1, and between DC-specific ICAM grabbing nonintegrin (DC-SIGN) and ICAM-2 and ICAM-3 (Geijtenbeek et al., 2000a
, b
). Finally, the fusion event should be mediated by the intervention of the as-yet-unidentified fusogenic factor encoded by the UL131128 locus. This mechanism should play its major role when infection occurs by co-culture of DC and endothelial cells. However, when cell-free virus infects DC, following extensive adaptation to HUVEC, the mechanism of infection could be similar, if UL131128 gene products are present in the viral envelope. The recently advocated role of DC-SIGN as the HCMV receptor that ultimately determines entry and replication of HCMV in permissive cells is not sufficient to justify virus entry into human fibroblasts, the most permissive cells, where DC-SIGN is not expressed (Halary et al., 2002
). Thus, DC-SIGN might be an important cellular co-factor mediating interactions of HCMV with DC and endothelial cells. These interactions can be initiated by virus attachment to heparan sulfate proteoglycans (Compton et al., 1993
). Subsequently, such an attachment may become stronger through virus interaction with annexin II, which binds to gB (Pietropaolo & Compton, 1997
) or a 92·5 kDa protein which binds to gH (Baldwin et al., 2000
) or, finally, DC-SIGN binding to gB. However, the final fusion event for entry of HCMV into leukocytes, DC and endothelial cells requires the intervention of a fusogenic factor, which could reasonably be represented by the UL131128 gene products. The amino acid sequence of the UL131, UL130 and UL128 products qualifies them as secretory proteins (Akter et al., 2003
; Hahn et al., 2004
), which could either physically or functionally interact with each other to trigger microfusion between adhering membranes of contiguous cells or even between viral envelope and cell membrane. Whether one of these proteins may be bound to viral envelope remains to be determined.
Our herein reported experimental findings indicate that infection of HUVEC with EC tropic HCMV strains at a high m.o.i. causes a striking syncytial effect within a few minutes or hours, which is totally absent when infection is attempted with a non-EC tropic virus strain inoculated at the same high m.o.i. (as determined on HELF). In addition, rapid penetration of the major component (pp65) of dense bodies (DB) from Huv+ Leuk+ viruses into the nucleus of HUVEC and DC was observed, whereas this finding was not observed when HUVEC (or DC) were incubated with DB containing virus preparations from Huv Leuk HCMV strains. Thus, both the syncytial effect and the nuclear targeting of pp65 within endothelial cells by Huv+ Leuk+ HCMV strains seem to suggest a fusion effect of both the virion and DB envelope with the cell membrane. This effect was not observed in UL131128 deletion mutants and in Huv Leuk viruses. Earlier work by Sinzger et al. (2000) reported that the block of AD169 (Huv Leuk) infection in HUVEC was a post-entry step involving translocation of viral DNA to the nucleus. Whether the lack of penetration of pp65 into the nucleus of HUVEC (and DC) may also be a post-entry event is not clear at the moment. Partial accumulation of pp65 inside the cytoplasm of DC as well as other cells was reported using purified DB preparations from AD169 infected human fibroblasts, but not following DC infection by an endotheliotropic HCMV strain (Pepperl-Klindworth et al., 2003
). However, using our virus preparations consisting of infected cell culture medium, we could not observe an accumulation of pp65 inside the cytoplasm of either HUVEC or DC, following incubation with Huv Leuk viruses. Finally, it must be kept in mind that the cell differentiation stage is critical for virus infection, as shown by the finding that monocytes cannot be productively infected unless previously differentiated into either macrophages (Ibanez et al., 1991
; Lathey & Spector, 1991
; Taylor-Wiedeman et al., 1991
; Minton et al., 1994
) or immature DC (Riegler et al., 2000
).
The use of DC infected with a wild-type virus appears to be a potent stimulator of both CD4+- and CD8+-mediated immune responses, if DC are co-cultured with PBMC within 24 h p.i. Subsequently, the antigenic stimulus is reduced, possibly due to the decreased expression of major histocompatibility complex (MHC) class I and II as well as co-stimulatory molecules and impaired pro-inflammatory cytokine production, thus resulting in impaired antigen presentation function (Andrews et al., 2001; Raftery et al., 2001
; Moutaftsi et al., 2002
). The study of the kinetics of the cell-mediated immune response following co-culture of PBMC with DC at different times from infection shows that CD4+ T-cell response has already occurred by 2 h p.i. and is close to the peak between 6 and 24 h p.i., whereas the CD8+-mediated immune response is delayed, reaching its peak at 24 h p.i. and then remaining stable between 24 and 48 h p.i.
The different kinetics of the CD4+- and CD8+-specific immune response allow us to speculate on the role of different viral proteins mostly contributing to induction of the T-cell immune response. The finding that the CD4+ anticipates the CD8+ T-cell response shortly after infection of DC may be due to the rapid penetration and processing of pp65 from DB with formation of pp65 peptides-MHC class II complexes, which are immediately transported and exposed on the cell membrane (Sallusto et al., 1995). Thus, presence in cytoplasmic vesicles of preformed MHC class II molecules and penetration of enveloped DB inside cytoplasmic vacuoles by phagocytosis may explain the earliness of the CD4+-mediated immune response. On the other hand, following penetration of DB with a mechanism shared by virions, pp65-derived peptides may be complexed to MHC class I molecules stimulating a CD8+ immune response. However, this process takes longer, since synthesis of MHC class I molecules is delayed (Rescigno et al., 1998
). In addition, at least in these experimental conditions, DC must be infected with endotheliotropic virus strains to generate the highest CD8+ T-cell response and peptides from de novo synthesized viral antigens other than pp65, such as IE1/2, may be exposed on the DC membrane during productive HCMV infection, thus increasing the overall level of the immune response. On the other hand, CD4+ T-cell activation is not substantially affected by the ability of the virus to infect DC. In this respect, it was also found that infection of DC with a DC-tropic virus blocked in its infectivity by neutralizing antibody strongly reduced only the CD8+ and not the CD4+ T-cell activation (data not shown).
The contribution of each of the major immunodominant proteins of HCMV to the cell-mediated immune response will be dissected in the near future by infecting DC with vaccinia virus recombinants individually carrying pp65, IE-1, gB and pp150 genes (Diamond et al., 1997; Gyulai et al., 2000
). Thus, the role played by each viral protein in eliciting CD4+ and CD8+ HCMV-specific immunity will be investigated in detail. However, the DC-based assay reported in this study allows a relatively rapid determination of both CD4+ and CD8+ T-cell immune responses using the same preparation of autologous infected DC. This may represent a major advance in the determination and follow-up of cell-mediated immune response of immunocompetent and immunocompromised patients. In fact, cytokine flow cytometry and a single blood sample can reliably evaluate both arms of the cell-mediated immune response.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Andrews, D. M., Andoniou, C. E., Granucci, F., Ricciardi-Castagnoli, P. & Degli-Esposti, M. A. (2001). Infection of dendritic cells by murine cytomegalovirus induces functional paralysis. Nat Immunol 2, 10771084.[CrossRef][Medline]
Baldwin, B. R., Zhang, C. O. & Keay, S. (2000). Cloning and epitope mapping of a functional partial fusion receptor for human cytomegalovirus gH. J Gen Virol 81, 2735.
Compton, T., Nowlin, D. M. & Cooper, N. R. (1993). Initiation of human cytomegalovirus infection requires interaction with cell surface heparan sulfate. Virology 193, 834841.[CrossRef][Medline]
Diamond, D. J., York, J., Sun, J. Y., Wright, C. L. & Forman, S. J. (1997). Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 90, 17511767.
Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H. & Liu, F. (2003). Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A 100, 1422314228.
Geijtenbeek, T. B., Krooshoop, D. J., Bleijs, D. A., van Vliet, S. J., van Duijnhoven, G. C., Grabovski, V., Alon, R., Figdor, C. G. & van Kooyk, Y. (2000a). DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking. Nat Immunol 1, 353357.[CrossRef][Medline]
Geijtenbeek, T. B., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Adema, G. J., van Kooyk, Y. & Figdor, C. G. (2000b). Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune response. Cell 100, 575585.[Medline]
Gerna, G., Revello, M. G., Percivalle, E., Zavattoni, M., Parea, M. & Battaglia, M. (1990). Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins. J Clin Microbiol 28, 26812688.[Medline]
Gerna, G., Percivalle, E., Baldanti, F., Sozzani, S., Lanzarini, P., Genini, E., Lilleri, D. & Revello, M. G. (2000). Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events. J Virol 74, 56295638.
Gerna, G., Piccinini, G., Genini, E. & 10 other authors (2001). Declining levels of rescued lymphoproliferative response to human cytomegalovirus (HCMV) in AIDS patients with or without HCMV disease following long-term HAART. J Acquir Immune Defic Syndr 28, 320331.[Medline]
Gerna, G., Percivalle, E., Baldanti, F. & Revello, M. G. (2002a). Lack of transmission to polymorphonuclear leukocytes and human umbilical vein endothelial cells as a marker of attenuation of human cytomegalovirus. J Med Virol 66, 335339.[CrossRef][Medline]
Gerna, G., Percivalle, E., Sarasini, A., Baldanti, F. & Revello, M. G. (2002b). The attenuated Towne strain of human cytomegalovirus may revert to both endothelial cell tropism and leuko- (neutrophil- and monocyte-) tropism in vitro. J Gen Virol 83, 19932000.
Gerna, G., Percivalle, E., Sarasini, A. & Revello, M. G. (2002c). Human cytomegalovirus and human umbilical vein endothelial cells: restriction of primary isolation to blood samples and susceptibilities of clinical isolates from other sources to adaptation. J Clin Microbiol 40, 233238.
Gerna, G., Baldanti, F., Percivalle, E., Zavattoni, M., Campanini, G. & Revello, M. G. (2003a). Early identification of human cytomegalovirus strains by the shell vial assay is prevented by a novel amino acid substitution in UL123 IE1 gene product. J Clin Microbiol 41, 44944495.
Gerna, G., Percivalle, E., Sarasini, A., Baldanti, F., Campanini, G. & Revello, M. G. (2003b). Rescue of human cytomegalovirus strain AD169 tropism for both leukocytes and human endothelial cells. J Gen Virol 84, 14311436.
Gyulai, Z., Endresz, V., Burian, K. & 7 other authors (2000). Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs. J Infect Dis 181, 15371546.[CrossRef][Medline]
Hahn, G., Khan, H., Baldanti, F., Koszinowski, U. K., Revello, M. G. & Gerna, G. (2002). The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild type characteristics. J Virol 76, 95519555.
Hahn, G., Revello, M. G., Patrone, M. & 9 other authors (2004). Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. J Virol 78, 1002310033.
Halary, F., Amara, A., Lortat-Jacob, H. & 7 other authors (2002). Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection. Immunity 17, 653664.[Medline]
Ibanez, C. E., Schrier, R., Ghazal, P., Wiley, C. & Nelson, J. A. (1991). Human cytomegalovirus productively infects primary differentiated macrophages. J Virol 65, 65816588.[Medline]
Lathey, J. L. & Spector, S. A. (1991). Unrestricted replication of human cytomegalovirus in hydrocortisone-treated macrophages. J Virol 65, 63716375.[Medline]
Minton, E. J., Tysoe, C., Sinclair, J. H. & Sissons, J. G. P. (1994). Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow. J Virol 68, 40174021.[Abstract]
Moutaftsi, M., Mehl, A. M., Borysiewicz, L. K. & Tabi, Z. (2002). Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells. Blood 99, 29132921.
Pepperl-Klindworth, S., Frankenberg, N., Riegler, S. & Plachter, B. (2003). Protein delivery by subviral particles of human cytomegalovirus. Gene Ther 10, 278284.[CrossRef][Medline]
Pietropaolo, R. L. & Compton, T. (1997). Direct interaction between human cytomegalovirus glycoprotein B and cellular annexin II. J Virol 71, 98039807.[Abstract]
Raftery, M. J., Schwab, M., Eibert, S. M., Samstag, Y., Walczak, H. & Schönrich, G. (2001). Targeting the function of mature dendritic cells of human cytomegalovirus: a multilayered viral defense strategy. Immunity 15, 9971009.[Medline]
Rescigno, M., Citterio, S., Thèry, C., Rittig, M., Medaglini, D., Pozzi, G., Amigorena, S. & Ricciardi-Castagnoli, P. (1998). Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells. Proc Natl Acad Sci U S A 95, 52295234.
Revello, M. G., Percivalle, E., Arbustini, E., Pardi, R., Sozzani, S. & Gerna, G. (1998). In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia. J Clin Invest 101, 26862692.
Revello, M. G., Baldanti, F., Percivalle, E., Sarasini, A., De-Giuli, L., Genini, E., Lilleri, D., Labò, N. & Gerna, G. (2001). In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leucocytes and to grow in endothelial cells. J Gen Virol 82, 14291438.
Riegler, S., Hebart, H., Einsele, H., Brossart, P., Jahn, G. & Sinzger, C. (2000). Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus. J Gen Virol 81, 393399.
Sallusto, F. & Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis factor . J Exp Med 179, 11091118.
Sallusto, F., Cella, M., Danieli, C. & Lanzavecchia, A. (1995). Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 182, 389400.
Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B. & Jahn, G. (2000). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81, 30213035.
Taylor-Wiedeman, J., Sissons, J. G. P., Borysiewicz, L. K. & Sinclair, J. H. (1991). Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol 72, 20592064.[Abstract]
Waldman, W. J., Knight, D. A., Huang, E. H. & Sedmak, D. D. (1995). Bidirectional transmission of infectious cytomegalovirus between monocytes and vascular endothelial cells: an in vitro model. J Infect Dis 171, 263272.[Medline]
Received 27 July 2004;
accepted 14 October 2004.