ballooning cells and giant syncytia was noted by day 5 p.i. in the PBL culture incubated in the ..... Yamanishi, M. Takahashi, and K. Baba. 1989. Seroepidemiol-.
Vol. 64, No. 9
JOURNAL OF VIROLOGY, Sept. 1990, P. 4598-4602
0022-538X/90/094598-05$02.00/0 Copyright C) 1990, American Society for Microbiology
T-Cell Activation Is Required for Efficient Replication of Human Herpesvirus 6 NIZA FRENKEL,1* ERIC C. SCHIRMER,1 GEORGE KATSAFANAS,1 AND CARL H. JUNE2
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases!Twinbrook, 12441 Parklawn Drive, Rockville, Maryland 20852,1 and Naval Medical Research Institute, Bethesda, Maryland 208142 Received 14 March 1990/Accepted 15 June 1990 We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host.
medium containing 32 U of interleukin-2 (IL-2) (purified natural human T-cell growth factor; Advanced Biotechnologies Inc., Silver Spring, Md.) per ml; and (iv) RPMI-10% medium containing both PHA and IL-2. The design of our studies was based on the fact that T-cell proliferation requires the binding of IL-2 to its high-affinity receptor (33, 37-39). Upon treatment of the cells with the polyclonal mitogen PHA, the IL-2 receptor is expressed. However, in the purified T-cell cultures, in the absence of accessory cells, IL-2 is not made and the cells are arrested in the G1 phase of the cell cycle. Thus, further progression in the cell cycle depends on the addition of exogenous IL-2. In contrast, in the unfractionated PBL, IL-2 is induced in the T cells in response to PHA stimulation. This results in full G1-to-S progression and cell proliferation (33, 37-39). Twenty-four hours after incubation in the various media, the cells were infected with cell-free HHV-6 (Z29) prepared from infected PBL. The mock-infected cultures were incubated with mock inoculum prepared in parallel with the virus stock from the same PBL used to prepare the HHV-6 inoculum. Induced cell proliferation in the various media was measured by counting the mock-infected cells 7 days after mock infection. The data (Table 1) revealed the expected effects on cell growth. Thus, only minimal cell proliferation was observed in the T-cell and PBL cultures incubated in medium alone or in medium containing IL-2 but no PHA. In the presence of PHA, the PBL cultures underwent extensive cell division. In contrast, the PHA-treated T cells did not replicate unless exogenous IL-2 was added to the medium. In an independent repeated experiment, these results were confirmed by [3H]thymidine incorporation assays (data not shown). Effects of cell activation on viral replication. Figure 1 shows representative photographs of the mock-infected and virusinfected cell cultures. No blastogenesis and no cytopathic effect (CPE) was apparent in the T-cell and PBL cultures cultured in medium only. Blast cells began to appear by day 7 in PBL cultured in medium containing IL-2. In contrast, the CD28+ purified T-cell cultures did not exhibit blastogen-
Human herpesvirus 6 (HHV-6) was originally isolated from patients with acquired immunodeficiency virus and from patients with lymphoproliferative disorders (2, 3, 11, 23, 34, 42). More recent seroepidemiological surveys have shown that the virus is ubiquitous in the human population and that seroconversion occurs early in life (7, 22, 32, 36, 40, 44, 46, 47). Infection ranges from subclinical illness (32, 36) to febrile illness and exanthem subitum, a common disease of children, characterized by high fever and skin rash (31, 40, 44, 47). Recent data have shown that HHV-6 infection promotes transcription from the human immunodeficiency virus long terminal repeat, and it has been suggested that it could play a role in activation of human immunodeficiency virus from latency (16, 25). HHV-6 exhibits a T-cell tropism (2, 11, 23, 26, 41), although at least one virus strain was found to replicate also in B cells and megakaryocyte and glioblastoma cell lines (1). We have investigated whether T-cell activation was required for HHV-6 replication by analyzing the growth requirements of the virus in unfractionated peripheral blood lymphocytes (PBL) and in purified CD28+ mature T cells. The PBL were prepared by centrifugation of buffy coats in Lymphocyte Separation Medium (Organon Teknika, Westchester, Pa.). CD28+ T cells were purified as previously described (19) by negative selection using immunoadsorption and elimination of cells expressing CD11, CD14, CD16, and CD20 surface antigens, thus removing macrophages, large granulocytes, natural killer cells, and B cells. CD28 is expressed in the majority of mature T cells, including 95% of the CD4+ and 50% of the CD8+ T cells (10). The unfractionated PBL and purified T cells were incubated in four types of medium promoting different degrees of cell proliferation. These corresponded to (i) RPMI-10% medium, consisting of RPMI 1640 medium, 10% fetal calf serum, and 50 Fig of gentamicin per ml; (ii) RPMI-10% medium containing 10 Lg of phytohemagglutinin (PHA P; Difco Laboratories, Detroit, Mich.) per ml; (iii) RPMI-10% *
Corresponding author. 4598
VOL. 64, 1990
Cells/ml (106)
None IL-2 PHA PHA + IL-2
T cells
PBL
1.32 1.46 1.37 5.27
1.19 1.43 4.91 6.05
a Cells were collected 7 days after mock infection in the presence of the medium as indicated. The number of cells was quantitated by using a counter
(Coulter Electronics).
esis after treatment with IL-2 alone. The delayed cell blastogenesis in the PBL cultured with IL-2 might have represented lymphokine activation of killer cells (17, 20, 24) or indirect activation of T cells in response to factors induced by IL-2 in accessory cells. Studies to further define the nature of these cells are currently in progress. No CPE was observed in the T cells cultured in the presence of IL-2 alone, whereas a few large ballooning cells, characteristic of HHV-6 CPE, were noted in the IL-2-treated infected PBL by day 7 postinfection (p.i.), coincidental with the observed cell blastogenesis. A pronounced CPE characterized by the appearance of
PBL
;tS*.,'^_3:
T
*o t,. .I
NTonet
IL-2
-
.9
4599
ballooning cells and giant syncytia was noted by day 5 p.i. in the PBL culture incubated in the presence of PHA. In contrast, no CPE was noted in the PHA-treated T-cell cultures. However, the T cells were infected when the cells were incubated in the presence of PHA and exogenous IL-2. Thus, infection of the cells under conditions that caused cell proliferation was accompanied by a characteristic viral CPE, whereas minimal or no CPE was detected in the cells cultured under conditions that did not promote efficient cell activation. Changes in cell size have been shown to correlate with transition from Go to G1 in the cell cycle of lymphocytes (33, 37-39). To quantify blastogenesis, the size distribution of the cells was determined by electronic sizing on day 7 p.i. Analyses of the mock-infected T cells (Fig. 2) revealed the presence of a distinct peak of larger cells in the culture treated with PHA plus IL-2. This peak was absent in the infected PHA- plus IL-2-treated T-cell culture. The absence of this peak resulted from fragmentation of the refractile infected cells which were visible by light microscopy and the concomitant generation of cell debris. The PBL cultures incubated with IL-2 alone, PHA, or with PHA plus IL-2 each contained a significant proportion of blast cells. As with the T cells, the population of blast cells, as determined by histograms of cell sizes, significantly decreased in the in-
TABLE 1. Cell counts in the presence of different mediaa Medium
NOTES
4 .
ii.
".
i,
PH A
.-
;
,
R
'
_;S .
_ ww .. ..
o.
....
_.s-'" ' ' '_>. C
wt r
b
.,
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{P
:
':
.>t
7t
NMock
Z29
I29 NItick and monitored in the text in daily intervals for viral CPE FIG. 1. Viral CPE in PBL or T-cell (T) cultures. Cells were infected as described with a light microscope. Representative photographs for day 5 p.i. are shown.
4600
J. VIROL.
NOTES
100
50
None
PHA
Mock
M
100
None
None
L
PHA -- -r
Moc
L
IHI JJ None PHA
PHA 4*
50
-
-_m
Z22
Z29L Z29
100 IL-2
50j
PHA+IL-2
i
Mock
Mock
L
IL-2
PHA+IL-2
Mock
Mock
I.-
IL-2
PHAk+IL-2
Z29
Z29
L
IL-2
PHA+IL-2
Z29
Z29
FIG. 3. Viral DNA in infected T-cell (T) and PBL cultures. Infected cell DNAs were extracted and hybridized as described elsewhere (13). To allow comparison, the lanes were loaded with equal amounts of DNA extracted from the infected cells. Lane 1 contains molecular size markers; sizes are in kilobases.
PIL T
PBL
FIG. 2. Size distribution of mock-infected and infected cells. Cultures were analyzed on day 7 p.i. in a counter (Coulter Electronics, Inc., Hialeah, Fla.) equipped with a channelyzer. T, T cells.
fected PBL cultures. These results suggest that cells undergoing blastogenesis represent the target for viral infection or virus-induced cell lysis or both. Infectious virus production in the PBL and T-cell cultures. The yields of infectious virus were determined on days 5 and 7 p.i. For virus titrations, 10-fold dilutions of the test stocks were mixed with PHA-treated PBL in RPMI-10% medium supplemented with 5 ,ug of PHA per ml. Infection was monitored by CPE and immunofluorescence. The results are summarized in Table 2. Only minimal amounts of virus were recovered from the T-cell and PBL cultures incubated in the absence of PHA and IL-2 and from the T-cell cultures treated with IL-2 alone. In contrast, virus production was noted in the PBL cultures treated with IL-2, reaching 104 infectious units (IU) per ml per 106 cells by day 7 p.i. The yield of infectious virus was highest in the T cells treated with PHA plus IL-2, reaching 106 IU/106 cells. Most notably, the yield of infectious virus was 2 to 3 logs lower in the T-cell cultures which were treated with PHA in the absence of exogenous IL-2, indicating that the addition of mitogen alone to T cells was not sufficient for optimal viral replication. Rather, optimal viral production correlated with conditions that cause cellular proliferation. Finally, high yields of infectious virus were attained in the PBL cultures incubated with PHA alone, as well as with PHA plus IL-2. It was also TABLE 2. HHV-6 replication in the presence of various mediaa Log IU/ml Medium
None IL-2 PHA PHA + IL-2
T cells
PBL
Day 5
Day 7
Day 5
Day 7
2 0 4 6
0 1 3 6
1 2 5 4
1 4 5 5
a Tenfold dilutions of the test cultures were incubated with fresh PBL. Virus presence was identified by the characteristic CPE. Infectious units are defined as the terminal dilution still producing infection and are expressed as infectious units per milliliter per 106 cells.
noted that in the cultures of PBL, the addition of IL-2 along with the PHA caused some delay in virus replication. The inhibitory effect was reproduced in additional experiments which showed that high concentrations of IL-2 caused significant inhibition of HHV-6 replication in thymocytes and PBL (33a; N. Frenkel, E. Roffman, E. C. Schirmer, G. Katsafanas, L. S. Wyatt, and C. H. June, in C. H. Lopez, B. Roizman, R. Mori, and R. J. Whitley, ed., Immunobiology and Prophylaxis of Human Herpesvirus Infections, in press). Specifically, recombinant IL-2 at concentrations higher than 10 U/ml or purified natural IL-2 at concentrations higher than 130 U/ml inhibited virus replication. The results concerning virus replication in the activated and nonactivated T-cell and PBL cultures were confirmed by using immunofluorescence assays (data not shown). Virus DNA replication in PBL and purified T-cell cultures. DNA extracted from the various cultures on day 5 p.i. was analyzed by Southern blot hybridization using pNF1001 which contained a 12-kilobase HindIII fragment of HHV-6 (Z29) DNA as a probe. Only small amounts of viral DNA were recovered from cultures in which cell replication did not occur (Fig. 3). Most notably, only low levels of viral DNA were recovered from T cells infected in the presence of PHA alone, whereas significant viral DNA replication occurred in the T cells cultured in medium containing PHA and exogenous IL-2. In the PBL culture, the addition of IL-2 along with the PHA was not critical, and significant DNA replication occurred in the presence of PHA alone. Finally, only limited amounts of viral DNA were recovered from the T-cell as well as PBL cultures which were infected in the presence of IL-2 alone by day 5 p.i. Taken together, the results of all assays quantifying virus replication revealed a positive correlation between cellular proliferation and virus replication. T-cell activation enhances viral replication. We have shown that HHV-6 replication in freshly isolated human T cells is enhanced following the induction of cell proliferation. In this respect, HHV-6 resembles human cytomegalovirus, as well as human immunodeficiency virus type 1 (5, 6, 14, 15, 18, 27, 28, 30, 35, 43, 48), the replication of which is enhanced by cell activation. Mitogenic activation and IL-2 addition have been routinely employed by investigators who propagate HHV-6 in
NOTES
VOL. 64, 1990
fresh lymphocytes, and in a recent study, Black et al. (4) have shown that the virus replicates better in cord blood lymphocytes cultured in the presence of PHA and IL-2 than in cultures not treated with PHA. However, quantitative correlation between cell proliferation and viral replication in pure T-cell cultures has not been reported. The mechanism by which T-cell activation affects HHV-6 replication is at present unknown. Resting cells would be refractory to HHV-6 infection if the virus utilizes surface protein(s) induced by cell activation as a specific receptor(s). Alternatively, the restriction could occur after entry into the cells, in the course of viral gene expression, viral DNA replication, and/or virion maturation. At present, we cannot exclude the first hypothesis, since the cell receptor for the virus has not been identified. However, a broader host cell range was reported for some HHV-6 strains (1, 11), making it unlikely that the viral receptor(s) is a surface protein expressed only after T-cell activation. Although information concerning the regulation of HHV-6 gene expression is as yet not available, it is reasonable to propose that one or more of the T-cell activation products could be involved in the control of HHV-6 gene expression, by analogy with human immunodeficiency virus type 1 (5, 15, 43) and human cytomegalovirus (18, 35). Furthermore, on the basis of our finding that only a low level of virus replication was observed in the PHA-treated T cells unless IL-2 was added to the culture, it can be suggested that the putative cellular function(s) necessary for efficient virus replication is induced during the IL-2-driven G1-to-S progression. Potential in vivo relevance. Regardless of the mechanism, it is reasonable to suggest that T-cell activation affects the outcome of in vivo HHV-6 infections by causing virus reactivation from a latent state and by affecting the severity and extent of disease when it occurs. Specifically, it has already been noted that the majority of children undergo seroconversion by the second year of life without apparent clinical disease (32, 36), whereas some of the infected children develop exanthem subitum or high fever without rash (40, 44, 46, 47). Furthermore, there is indirect evidence in support of the hypothesis that HHV-6 remains latent in the host and can be reactivated from this state. Thus, peripheral blood cells were shown to harbor HHV-6 genomes detectable by the polymerase chain reaction (8). The virus was recovered from patients with acquired immunodeficiency syndrome (2, 3, 11, 23, 34, 42), and lymphoid cells positive for HHV-6 antigens were detected in cervical lymph nodes of healthy individuals (9, 12, 21, 31). Self-limiting febrile illness coupled with seroconversion was found in renal transplant recipients (29, 45). Finally, recent evidence from our laboratory has shown that HHV-6 can be reactivated in vitro from cultures of PBL from healthy individuals (N. Frenkel, G. Katsafanas, E. C. Schirmer, and L. S. Wyatt, unpublished data). One might hypothesize that coincidental stimulation of T cells by a nonrelated bacterial infection or a different viral infection(s) might play a role during putative viral activation from latency and might enhance the immunopathogenicity of primary HHV-6 infections. In this respect, it is noteworthy that we have recently isolated a new human herpesvirus, HHV-7, from CD4+ T cells of a healthy individual (13). HHV-7 was apparently induced in the course of T-cell proliferation. It remains to be seen whether T-cell stimulation in vivo plays a role in virus reactivation from latency and whether it affects primary virus replication and the severity of disease.
4601
We thank Nancy Craighead for excellent technical assistance in purification of the CD28+ cells and Dario Di Luca and Charlotte J. Steele for preparation of the pNF1001 probe. LITERATURE CITED
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