XMRV replication is restricted in CEM cells but not in CEM-SS cells.
The protocol used to compare the kinetics of replication and spread of XMRV in A3G/A3F-expressing CEM and A3G/A3F-deficient CEM-SS cells is outlined in A. Infectious XMRV was harvested from prostate carcinoma cell line 22Rv1. The 22Rv1 cell line has been shown to produce large amounts of a virus that is virtually identical to XMRV, originally found in prostate cancer samples (22
). We quantified the amount of XMRV harvested from the 22Rv1 cell line by using real-time RT-PCR to detect XMRV env
RNA as previously described (20
). The virus stock was diluted, and different amounts of XMRV RNA copies (104
, or 1010
) were used to infect 1 × 106
CEM or CEM-SS cells. Culture supernatants from the infected cells were collected every day for 14 days, and the XMRV particles present were quantified using real-time RT-PCR.
Fig. 1. Replication of XMRV in CEM and CEM-SS cells. (A) Schematic overview of the experimental protocol. Supernatant containing XMRV was harvested from 22Rv1 cells, and XMRV RNA copies in the supernatant were determined by quantitative real-time RT-PCR using (more ...)
The amounts of XMRV produced from the infected CEM and CEM-SS cells are shown in B to G. Neither CEM nor CEM-SS cells infected with 22Rv1 culture supernatant containing 104 RNA molecules produced detectable amounts of XMRV (detection limit, ~1.1 × 102 copies/8.4 μl) (B). XMRV particles were detected in both CEM and CEM-SS cells infected with 105, 106, 107, 108, or 1010 viral RNA copies (C to G, respectively); virus production at the beginning of infection (days 1 to 3) was dependent on the amount of XMRV used to infect the cells, and there was no difference in the virus production between CEM and CEM-SS cells (B to G).
Virus production increased to detectable levels in infected CEM-SS cells starting around days 5 to 7, whereas viral production either did not increase or increased only slightly in CEM cells infected with 105 to 1010 XMRV RNA copies (C to G). Virus production did not increase in either CEM-SS or CEM cells infected with 104 RNA copies, indicating that exposure to culture supernatant containing 104 XMRV RNA copies did not result in an infection event (B). In contrast, while no virus production could be detected in CEM cells infected with 105 RNA copies, infection was clearly established in the CEM-SS cells, as evidenced by the dramatic increase in virus production from less-than-detectable levels at day 6 to 2.5 × 105 copies per 8.4 μl at day 14 (C). A similar increase in virus production was observed in CEM-SS cells infected with 106, 107, and 108 RNA copies, whereas less than a 10-fold increase was observed in CEM cells (D, E, and F). In CEM-SS cells infected with 1010 RNA copies, a modest 18-fold increase in virus production was observed, and no increase was observed in CEM cells (G).
To compare the increases in virus production, we compared the average number of viral RNA copies in culture supernatants from days 1 to 3 and days 12 to 14 to determine the increase in RNA copies (H). The results showed a remarkable increase in virus production in CEM-SS cells infected with 105, 106, 107, and 108 RNA copies; the increase in virus production ranged from ~863-fold to ~4,276-fold. In contrast, there was only a modest increase in virus production in CEM cells, which reached a maximum of ~14-fold in cells infected with 108 RNA copies. Only an ~18-fold increase in virus production was detected in CEM-SS cells infected with 1010 RNA copies, suggesting that a large proportion of the cells were infected with the input virus, and a plateau was reached after all the cells were infected, resulting in superinfection interference. This result is consistent with the estimation that exposure of CEM or CEM-SS cells to 105 RNA copies results in 1 to 10 infection events (compare B and C). Thus, infection with 1010 RNA copies of input virus would result in up to 1 × 106 infection events, and a large proportion of the 1 × 106 CEM-SS cells would be infected with input virus; consequently, most of the cells would be infected after a small increase in virus production, and superinfection interference would be established, suppressing any additional increase in virus production.
An increase in virus producer cells does not account for the increase in virus production.
To determine whether an increase in virus producer cells could have contributed to the modest (~14-fold) increase in virus production observed in CEM cells (H), we repeated the experiment described for and quantified the virus producer cells every 2 days. The fold increase in virus production was determined by dividing the average RNA copies present on days 13 to 15 with the average RNA copies present on days 1 to 3 (A). The results were similar to those observed in H; the increase in viral production ranged from ~6- to ~3,936-fold in CEM-SS cells and up to 34-fold in CEM cells. In this experiment, the increase in viral load in CEM-SS cells after infection with 105 RNA copies was 77-fold, compared to 1,050-fold in H. Since <10 cells were likely to be infected after infection with 105 RNA copies, the variability in viral production may be due to stochastic events that led to greater variation in the number of initially infected cells or loss of virus production from some of the initially infected cells.
Fig. 2. Increase in virus production and proviral copy numbers in infected CEM and CEM-SS cells. (A) CEM and CEM-SS cells were infected as described for A, and the increase in virus production was determined as described for H. (B) The increase in (more ...)
To determine the extent to which the number of virus-producing cells affected virus production, the increases in virus production were adjusted for the numbers of cells in culture (B). The increase in virus production in CEM and CEM-SS cells was generally reduced by 2- to 4-fold after adjusting for the number of virus-producing cells, but the CEM cells still exhibited modest increases in virus production (up to 10-fold). Therefore, despite the presence of A3G and A3F, XMRV replication was modestly increased in CEM cells infected with 106, 107, and 108 RNA copies.
We also analyzed the extent of XMRV replication in CEM and CEM-SS cells by isolating genomic DNA from day 3 and day 13 after infection and determining the number of XMRV proviral copies present by quantitative real-time PCR (C). The proviral copies per cell were very low at day 3 in all infections except in infections with 1010 RNA copies and were greatly increased at day 13, reaching a maximum of ~10 in CEM-SS cells. In both CEM and CEM-SS cells infected with 1010 RNA copies, the proviral copies per cell were ~2 at day 3 and day 13.
The fold increase in proviral copy numbers was determined by comparing the proviral copies per cell present at day 3 and day 13 (D). In CEM-SS cells, the proviral copies per cell increased 14-, 960-, 490-, and 32-fold after infection with 105, 106, 107, and 108 RNA copies, respectively, which were generally similar to the increases in virus production (B and D, compare light gray bars). Proviral copy numbers per cell did not significantly increase in CEM and CEM-SS cells infected with 1010 RNA copies, suggesting that nearly all of the cells were infected by day 3, and superinfection interference was established. In CEM cells, the proviral copies per cell increased 14-, 22-, and 12-fold in cells infected with 106, 107, and 108 RNA copies, respectively. In these cells, virus production increased 4.7-, 9.6-, and 7.1-fold, suggesting that the increase in proviral copies per cell was ~2- to 3-fold greater than the increase in virus production. This result is consistent with the expectation that most of the proviruses in the CEM cells would be hypermutated and would be unable to produce viral particles.
XMRV replication is severely restricted in PHA-activated human PBMCs.
Since XMRV replication and spread was restricted in A3G/A3F-expressing CEM cells, we sought to determine the extent to which XMRV can replicate and spread in A3G/A3F-expressing human PBMCs. We first compared the steady-state levels of A3G and A3F proteins in PHA-activated PBMCs obtained from three different donors to the levels present in human T cell line and prostate cancer cell lines (A). As expected, A3G and A3F expression was clearly detected in CEM and H9 cells but not CEM-SS cells, and A3G was not detectable in prostate cancer cell lines 22Rv1, LNCaP, and DU145. Compared to CEM cells, A3F expression was similar in H9 cells and substantially lower in LNCaP, 22Rv1, and DU145 cells. A3G and A3F were expressed in PBMCs from all three donors at levels similar to or higher than those in CEM cells.
Fig. 3. Replication of XMRV in PBMCs. (A) A3G and A3F expression in T cell lines (CEM, CEM-SS, and H9), prostate cancer cell lines (22Rv1, LNCaP, and DU145), and PBMCs from three different donors. Western blotting employed an equal amount of protein (10 μg); (more ...)
To determine the extent of XMRV replication and spread in A3G/A3F-expressing PBMCs, 22Rv1 culture supernatants containing 105, 106, 107, 108, and 1010 XMRV RNA copies were used to infect 1 × 107 PBMCs from donor 2 (B). Consistent with the results obtained with CEM cells, no virus was detected in PBMCs infected with culture supernatants containing 105 XMRV RNA copies, suggesting that this amount of virus is not sufficient to establish a productive infection in PBMCs. In culture supernatants of PBMCs infected with 106 XMRV RNA copies, a low level of virus was detected, which became undetectable after 7 days. In culture supernatants of PBMCs infected with 107, 108, and 1010 XMRV RNA copies, detectable levels of virus were present early in infection, which ranged from 2 × 102 to 2 × 105 RNA copies per 8.4 μl of culture supernatant. Importantly, we did not observe any increase in viral RNA copies in the infected PBMCs during the next 15 days, indicating that there was little or no virus replication and spread. To quantify viral replication and spread, we compared the average RNA copy numbers from days 1 to 3 and days 13 to 15 (C). Viral RNA copy numbers did not increase, since the ratios of the viral RNA copy numbers on days 13 to 15 to days 1 to 3 were less than 1 (0.14 to 0.5), indicating that XMRV replication and spread in activated human PBMCs is severely restricted.
We also compared the proviral copy numbers per cell in PBMCs infected with 1010 XMRV RNA copies from day 1 and day 15 (D). The proviral copy numbers per cell in the three donors on day 1 averaged ~0.07, compared to ~2 proviruses per CEM cell after infection with 1010 XMRV RNA copies; since 10-fold more PBMCs (1 × 107) were infected compared to CEM cells (1 × 106), infection of PBMCs was ~3-fold less efficient (~2 × 106 proviruses in CEM cells versus ~7 × 105 proviruses in PBMCs). The proviral copy numbers per cell did not increase in the PBMCs from day 1 to day 15 and actually decreased from 0.07 to 0.03 proviral copies/cell, but the decrease was not statistically significant (P = 0.15; t test); these results indicated that XMRV replication and spread is severely restricted in PBMCs. Viral production from the PBMCs was approximately 10-fold lower than in CEM cells, which is in general agreement with the ~3-fold reduction in infection efficiency.
Murine leukemia viruses require cell division to integrate into the target cell chromosomes and complete viral replication (25
). To determine whether XMRV replication was restricted in PBMCs because of inefficient cell division, we infected PBMCs on days 1, 5, and 9 after activation and monitored virus production (E). Similar virus production was observed after infection on all 3 days, indicating that equivalent numbers of cells on days 1, 5, and 9 were susceptible to XMRV infection. Thus, the severe restriction to virus replication and spread in the PBMCs was not because of the absence of cells susceptible to XMRV infection.
Hypermutation of XMRV proviruses in PHA-activated PBMCs.
To analyze the effects of A3G/A3F expression on XMRV replication in PBMCs, we harvested XMRV-infected cells at day 15, isolated total cellular DNA, PCR amplified and cloned a 1.2-kb fragment from the pol region of XMRV proviral DNA, and sequenced the cloned PCR products (). Proviral sequences were recovered from PBMC cultures infected with 1010 RNA copies from the three different donors described for ; of the 38, 40, and 46 sequences obtained at day 15 from donors 1, 2, and 3, respectively, 2, 6, and 18 (~5 to 39%) sequences were hypermutated. Furthermore, most of the G-to-A substitutions occurred in the GG dinucleotide context, which is characteristic of A3G-mediated hypermutation. The detection of these hypermutated proviruses in PBMCs indicated that some of the PBMCs were infected with virus that was produced from A3G-expressing cells. A few of the sequences also had multiple G-to-A mutations in the GA dinucleotide context, which is characteristic of A3F-mediated hypermutation. These results indicated that XMRV produced from the PBMCs packaged A3G or A3F into the virions and reinfected the PBMCs, resulting in G-to-A hypermutation in the context of GG or GA dinucleotides, respectively.
Fig. 4. G-to-A hypermutation of XMRV proviral DNA in PBMCs. PBMCs infected with XMRV containing 1010 RNA copy numbers were harvested at day 15 from three different donors, and genomic DNA was isolated. A 1.2-kb fragment (nucleotides 2465 to 3660) of the viral (more ...) A sensitive assay for detection of replication-competent virus in XMRV-infected cells.
To determine whether infectious XMRV can be recovered from infected PBMCs or from other human cells, we developed a sensitive assay for recovery and detection of replication-competent XMRV. The strategy for XMRV detection and the construction of the DIG cells is illustrated in A. D17 canine osteosarcoma cells were transfected with the previously described MLV-based plasmid pMS2-FP-GF-no3R. This vector contains modified LTRs, in which partially redundant portions of the gfp gene are located in each LTR such that the gfp gene is reconstituted upon reverse transcription. The plasmid includes the MLV packaging signal (Ψ), the hygromycin phosphotransferase B gene (hygro), which is expressed from the SV40 promoter, and the FP portion derived from the 3′ end of gfp in the left LTR and the GF portion derived from the 5′ end of gfp in the right LTR. In addition, the vector contains all other cis-acting sequences required for retroviral replication but lacks gag, pol, and env, and thus can only be packaged by a helper virus that has infected the same cell. XMRV and MLV packaging sequences (Ψ) share ~70% homology, and XMRV has previously been shown to package MLV-based vectors. During reverse transcription of the MS2 vector, the middle F portion of gfp, which is present in both LTRs and has replaced the R region of the MLV LTR, is used to carry out the minus-strand DNA transfer step of reverse transcription. This results in functional reconstitution of the gfp gene and expression of functional GFP. A D17 cell line that was constructed to stably express the MS2 vector was named the DIG cell line. When XMRV infects DIG cells, many of the newly assembled XMRV particles will package the DIG RNA instead of XMRV RNA, resulting in GFP-positive cells in subsequent rounds of infection.
Fig. 5. Characterization of DIG cells and isolation of replication-competent XMRV from infected PBMCs. (A) Structure of MLV-based vector pMS2. FP, the 3′ 462-bp fragment of GFP; GF, the 5′ 350-bp fragment of GFP; Ψ+, the extended MLV packaging (more ...)
The protocol used to demonstrate the sensitivity of XMRV detection using the DIG cells is outlined in B. Briefly, we infected the DIG cells with culture supernatants from 22Rv1 cells containing 6 × 103
to 6 × 108
RNA copies. After 5 h, the virus-containing medium was removed, and the cells were washed with fresh medium. The infected cells were further incubated for 12 to 16 h, at which point 3 × 103
LNCaP cells were added (day 1), to ensure that highly infectible cells were present in the culture. Other experiments have indicated that the D17 cells can be efficiently infected with XMRV (14
) (data not shown). The infected cells were monitored every day for 5 days (days 2 to 6) by using high-content imaging (ImageXpress; Molecular Devices), and the percentage of GFP-positive cells was determined. A representative result of three independent experiments (C) shows that XMRV replication and spread were detectable in all cultures that were infected with XMRV.
Recovery of replication-competent XMRV from infected PBMCs.
To detect replication-competent XMRV from infected PBMCs, we cococultured DIG cells with PBMCs that were infected with different amounts of XMRV and monitored GFP expression in the DIG cells after 14 and 25 days (D). Activated PBMCs (6 × 106) from donor 2 were infected with different amounts of XMRV RNA copies, as described earlier (), and virus replication was monitored (E). Similar to donor 2 (B), no increase in virus production was observed after 8 days (E). In fact, the virus production slightly decreased over the course of the experiment, which corresponded with a decrease in the total number of live cells in the cultures. Virus production in the PBMCs infected with 105 RNA copies decreased to undetectable levels 5 days after infection. At 7 days postinfection, 1 × 105 infected PBMCs were cocultured with 8 × 105 DIG cells for 25 days. After 14 days (data not shown) and 25 days, DIG cells were harvested and the percentage of GFP-positive cells was determined by FACS analysis (F). Additional studies are required to determine the minimum amount of time required for recovery of infectious virus using the DIG cells. PBMCs infected with 1010, 108, and 107 XMRV RNA copies generated XMRV that infected the DIG cells. Subsequent spread of the MLV reporter vector in the DIG cells increased the proportion of GFP-positive cells to 31 to 41%. However, infectious XMRV could not be recovered from PBMCs infected with 104, 105, or 106 XMRV RNA copies.