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Keywords:

  • COS-tat;
  • HIV-1 Tat;
  • progressive multifocal leukoencephalopathy-type JC virus

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. REFERENCES

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.

List of Abbreviations: 
AIDS

acquired immunodeficiency syndrome

COS-tat

COS-7-derived cell lines stably expressing HIV-1 Tat

CPE

cytopathic effect

EMEM

Eagle's minimum essential medium

HA

hemagglutination

HAU

HA units

HIV-1

human immunodeficiency virus type 1

JCV

JC virus

MTT

3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl tetrazolium bromide

PML

progressive multifocal leukoencephalopathy

PML-type JCV

JCV isolated from the brain of PML patients

Purα

purine-rich element binding protein A

SV40 ori

simian virus 40 origin

TAR

trans-acting responsive region

JC virus is a causative agent of PML, a fatal demyelinating disease of the central nervous system in immunosuppressed patients (1). The high incidence of PML among individuals with AIDS in comparison with other immunocompromised patients implies that the presence of HIV-1 in the brains of infected individuals is closely associated with the pathogenesis of AIDS-related PML. It is known that HIV-1 encodes Tat protein, which is a potent trans-activator essential for virus transcription (2). Tat protein is detected in both infected cells and uninfected oligodendrocytes in the brains of AIDS patients (2). Previous reports have shown that HIV-1 Tat protein increases the basal activity of the JCV late promoter and that the trans-acting responsive region-homologous sequence of the JCV genome is essential for this process (3, 4). It is also known that a cellular protein, Purα, and Tat act together to stimulate DNA replication initiated at the JCV origin (5–7). From these lines of evidence, it is thought that the high incidence of PML in AIDS patients is related to Tat-mediated activation of JCV propagation in the brain. Previously, we established several COS-7-derived cell clones which stably express HIV-1 Tat (COS-tat cells) (8). In this previous study, we found that stable expression of Tat results in increased replication of non-pathogenic JCV with archetype regulatory regions of the viral genome, and that the efficiency of JCV propagation in COS-tat cells is related to the degree of Tat activity (8). However, archetype JCV has not been implicated as an etiologic agent of PML (9–11), and it is unknown whether stable expression of Tat promotes propagation of PML-type JCV with a hypervariable regulatory region of the viral genome. In this study, we have examined the propagation characteristics of PML-type JCV in COS-tat cells.

COS-tat cell lines were established by the transfection of COS-7 cells with HIV-1 Tat expression plasmid (8). Although COS-tat cells can be cultivated under normal culture conditions, we have found that these cells exhibit different morphologies and proliferate slowly compared to parental COS-7 cells (S. Nukuzuma, unpublished data). Proliferation characteristics of COS-tat cells may provide important background information for studies using these cell lines. Thus, we first compared the cell proliferation of three COS-tat cell lines with those of parental COS-7 cells. COS-7 cells (ATCC CRL 1651) and COS-tat cell clones (8) were cultivated in EMEM containing 10% FBS (hereafter called culture medium). Cell cultures were maintained at 37°C in a humidified incubator containing 5% CO2 in air. The relative number of live cells was determined by measuring mitochondrial succinate dehydrogenase activity using MTT assay. COS-7 cells and COS-tat cell clones were each plated in five wells of 96-well culture plates at a concentration of 2 × 103 cells/well in 100 μL culture medium and incubated at 37°C in a CO2 incubator. MTT assay was performed using a Cell Proliferation Kit I (MTT) (Roche, Penzberg, Germany) according to the manufacturer's instructions. After an incubation period of 5 days, 10 μL MTT solution was added to each well to a final concentration of 0.5 mg/mL, and the plates incubated for 4 hr. Then, 100 μL solubilization solution was added to each well, and the plates placed in an incubator overnight. The formazan products were solubilized, and spectrophotometric data were measured using an enzyme-linked immunosorbent assay reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 550 nm with a reference wavelength of 650 nm. The significance of inter-group differences was statistically determined by Student's t-test. As shown in Table 1, the enzyme activity of COS-tat7 and COS-tat15, and COS-tat22 cells was lower than that of parental COS-7 cells and this difference was statistically significant (P < 0.01). Of note, the enzyme activity of COS-tat22 cells was lower than that of COS-tat7 and COS-tat15 cells (P < 0.01). To measure the doubling time, COS-7 cells and COS-tat cell clones were plated in 6-well culture plates at a concentration of 4 × 104 cells/well in 2 mL culture medium. After an incubation period of 72 hr, cell numbers were counted. The doubling time of COS-7, COS-tat7, COS-tat15, and COS-tat22 were 21.6, 24.6, 22.8, and 30.8 hr, respectively. The doubling time of COS-7 COS-tat cells were in agreement with the proliferation characteristics of the cells as judged by MTT assay. Taken together, these results indicate that stable expression of Tat leads to down-regulation of cell proliferation.

Table 1.  Proliferation characteristics of COS-tat cell clones and parental COS-7 cells††
CellsAbsorbance‡
  1. ††, COS-7 cells and COS-tat cell clones were incubated for 5 days, and cell proliferation was measured by the MTT assay, as described in the text.

  2. ‡ Data are shown as the means ± standard deviations of the means (n= 5 per group); *, P < 0.01 (COS-7 versus COS-tat7, COS-tat15, and COS-tat22); **, P < 0.01 (COS-tat22 versus COS-tat7 and COS-tat15).

COS-70.326 ± 0.013
COS-tat70.285 ± 0.010*
COS-tat150.296 ± 0.009*
COS-tat220.233 ± 0.006*,**

We next compared the production of PML-type JCV in COS-tat cell clones with that in parental COS-7 cells. Since JCV capsids have the property of agglutinating human type O erythrocytes, HA assay has been traditionally employed to determine the virus titer (12). COS-7 and COS-tat cell clones were cultured in 35-mm dishes containing 2 mL culture medium until the cells were 50–80% confluent. Genomic DNA of PML-type JCV (Mad-1/CR-JCI) (13) was used for the assay of virus production. Transfection experiments were carried out essentially as described previously (8). Briefly, viral DNA (1.5 μg/culture) was excised from recombinant plasmid and introduced into the cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA). Thereafter, the transfected cells were transferred into 25-cm2 flasks containing culture medium and passaged at a split ratio of 1:3 or 1:4 every 3 or 4 days. Cells were harvested at 30, 43, and 50 days after transfection, and the HA titer was determined as described previously (8, 14). Experiments were performed using four independent cultures. The transfected cells exhibited no obvious CPE and were able to be passaged serially for 3 weeks of incubation. Thirty days after transfection, obvious CPE (rounding of the cells) was observed in a small population of all COS-tat cell clones (data not shown). The cells were subjected to HA assay at 30, 43, and 50 days after transfection. At 30 and 43 days after transfection, HA titers of COS-tat cell clones were significantly greater than those of parental COS-7 cells (Fig. 1a, b). In COS-tat7 cells, HA titer peaked at 43 days and remained unchanged up to 50 days after transfection (Fig. 1a–c). HA activity in COS-tat15 cells increased gradually from 30 to 50 days, with a peak at 50 days after transfection (640 ± 256 HA units) (Fig. 1a–c). HA activity in COS-tat 22 cells increased steeply up to 30 days compared to that in other COS-tat cell clones (Fig. 1a) and was similar to that in parental COS-7 cells at 50 days after transfection (Fig. 1c). These results indicate that stable expression of HIV-1 Tat leads to increased production of PML-type JCV in COS-tat cells. The data also suggest that the kinetics of PML-type JCV propagation differ among COS-tat cell clones.

image

Figure 1. Production of viral particles in COS-tat cell clones and parental COS-7 cells. Cells were transfected with genomic DNA of PML-type JCV. At (a) 30, (b) 43, and (c) 50 days after transfection, the cells were harvested, and HAU of total yield were determined as described in the text. In untransfected cells, HA activity was under the detectable level (<2 HAU). Data are shown as the means ± standard deviations of the means (n= 4 per group).

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To confirm HIV-1 Tat-mediated propagation of PML-type JCV, we examined the replication of viral genomic DNA in COS-tat cell clones. Total DNA was isolated from the above-mentioned HA samples using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and subjected to real-time PCR analysis for quantification of JCV genomic DNA, essentially as described previously (8, 14). The detectable range of real-time PCR was more than 100 copies per reaction in this system (8, 14). The amount of viral DNA in COS-tat7, COS-tat15, and COS-tat22 cells was significantly greater than that in parental COS7 cells at 30 days after transfection (Fig. 2a). In COS-tat7 cells, viral DNA level peaked at 43 days after transfection and declined at a later time point (Fig. 2b, c). The amount of viral DNA in COS-tat15 cells gradually increased from 30 to 50 days after transfection (Fig. 2a–c). In COS-tat22 cells, the amount of viral DNA increased steeply up to 30 days after transfection compared to other COS-tat cell clones. Although COS-tat22 cells exhibited a steep increase in the amount of viral DNA compared to other COS-tat cell clones on day 30, the amount decreased from 43 to 50 days (Fig. 2a–c). These results indicate that stable expression of HIV-1 Tat in COS-tat cells facilitates replication of PML-type JCV. The data also suggest that the replication kinetics of PML-type JCV DNA differ among COS-tat cell clones.

image

Figure 2. Replication of JCV genomic DNA in COS-tat cell clones and parental COS-7 cells. Cells were harvested at (a) 30, (b) 43, and (c) 50 days after transfection with viral DNA, as described in the legend to Figure 1. Total DNA was extracted from HA samples and the amount of JCV DNA in each sample (20 μL HA sample) was quantified by real-time PCR, as described in the text. The amount of JCV DNA in each sample was normalized with reference to the copy numbers of simian β-actin DNA. In untransfected cells, JCV DNA was under the detectable level (100 copies/reaction). Data are shown as the means ± standard deviations of the means (n= 4 per group).

Download figure to PowerPoint

In the current study, we examined the propagation characteristics of PML-type JCV in COS-7 derived cell lines expressing HIV-1 Tat protein. In COS-tat cells, production of virus progenies and replication of viral genomic DNA were increased compared to those in parental COS-7 cells, as judged by data from HA and real-time PCR assays. Based on the results obtained in the present and previous studies (8), we have demonstrated that stable expression of HIV-1 Tat facilitates propagation of, not only archetype, but also PML-type, JCV. In COS-tat cells, HIV-1 Tat-mediated JCV propagation can be examined without transfecting the cells with Tat expression plasmid or stimulating them with exogenous Tat. Thus, these cell lines may provide a useful model system for studying HIV-1 Tat-mediated propagation of both archetype and PML-type JCV.

When examining the characteristics of COS-tat cells, we found that stable expression of HIV-1 Tat resulted in down-regulation of cell proliferation. This reduction of the cell growth of COS-tat cells is consistent with earlier results indicating that Tat prevents proliferation of human intestinal epithelial cells (15). A growing body of evidence suggests that HIV-1 Tat regulates numerous cellular genes that are involved in cell signaling and translation, thereby controlling the proliferation of host cells (16). The precise mechanism by which Tat protein represses the proliferation of COS-tat cells is unclear; however, previous investigations suggest that HIV-1 Tat induces the expression of Purα, a single-stranded DNA binding protein which inhibits cell growth (16, 17). Therefore, it might be that the decreased proliferation of COS-tat cells is associated with Tat-induced expression of Purα.

In our previous study, archetype JCV efficiently propagated in COS-tat7, COS-tat15, and COS-tat22 (8). Among the COS-tat cell clones tested, COS-tat22 cells exhibited a marked increase in the propagation of archetype JCV at about 30 days after transfection with viral DNA (8). Consistent with earlier results, amounts of HA and viral DNA in COS-tat22 cells were greater than those in other COS-tat cell clones at 30 days following transfection with PML-type JCV DNA. It is likely that production of Tat protein leads to increased propagation of archetype and PML-type JCV in three COS-tat cell clones, although the extent of its expression varies between these clones (8). It has been reported by others that Tat protein can enhance late-promoter transcription of JCV through interaction with a sequence similar to TAR in the JCV control region (3, 4). It has also been demonstrated that Tat protein forms a complex with Purα, thereby stimulating viral DNA replication initiated at the JCV origin (5, 6). Thus, it is probable that Tat protein facilitates JCV propagation in COS-tat cells via a Purα-dependent mechanism.

We also found that COS-tat15 cells showed a significant increase in HA activity and the amount of viral DNA at later time points (43 and 50 days) compared to COS-tat22 cells. These results suggest that COS-tat15 cells continuously produce JCV progenies in long-term culture. The reason for the different kinetics of JCV propagation between COS-tat15 and COS-tat22 cells is currently unclear; however, our previous data indicate that Tat activity in COS-tat15 cells is lower than that in COS-tat22 cells (8). A previous study demonstrated that maximum stimulation by Tat protein occurs at low concentrations (about 10−7 M) and declines at higher ones (7). Thus, it is likely that, although Tat promotes JCV propagation, excessive Tat activity may not be necessary for promotion of JCV propagation in COS-tat15 cells at later time points (43 and 50 days).

Stable expression of Tat is an important feature for generating JCV propagation system using COS-tat cells. The Tat-expression plasmid (pcDNA-tat86) contains SV40 ori and is able to replicate in COS-7-derived cells expressing SV40 T antigen. This may be associated with constant expression of HIV-1 Tat protein in COS-tat cell clones during long-term culture, while it is also likely that the Tat-expression construct is integrated into the host cell chromosome. However, we cannot totally exclude the possibility that long-term culture leads to an alteration in the characteristics of COS-tat cells. However, in the preliminary experiments, the growth characteristics and cell morphologies of COS-tat cells seemed not to be affected by long-term culture (data not shown). Further analyses, such as profiling of Tat and host gene expression, need to be conducted to better understand Tat-mediated JCV propagation in COS-tat cells during long-term culture.

In conclusion, the data obtained in the current study demonstrate that stable expression of HIV-1 Tat increases propagation of PML-type JCV. To our knowledge, the results of the present study constitute the first demonstration of increased propagation of PML-type JCV in long term-culture of cell lines stably expressing HIV-1 Tat.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. REFERENCES

We thank Hyogo Red Cross Blood Center for kindly providing human O type blood for HA assay. This work was supported by Grants-in-Aid from the Research Committee of Prion Disease and Slow Virus Infection, the Ministry of Health, Labor and Welfare of Japan, and in part by a Grant for Project Research from the High-Tech Center (H2010-10) of Kanazawa Medical University.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. ACKNOWLEDGMENTS
  4. REFERENCES
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