Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

The rationale for focusing on FeLV-B as the most likely zoonotic variant is presented in Fig. 1A. FeLV-B is the most commonly occurring envelope variant with human cell tropism and is formed by recombination with endogenous FeLV-related sequences (20) (Fig. 1B). To generate FeLV-B virus stocks, the prototypic molecular clone of Gardner-Arnstein FeLV-B was reconstituted by transfection, propagated in HEK293 cells, which release high-titer FeLV-B into cell supernatants (10⁵ to 10⁶ infectious units/ml), and titrated on feline QN10S cells (33). In initial experiments, it was noted that human cells in culture were invariably susceptible to virus infection, but with markedly different proviral copy numbers as assessed by Southern blotting or quantitative PCR. This phenomenon was explored further in a dilution/spreading assay where cells were exposed at decreasing multiplicities of infection (MOI) (from 1 to 10⁻⁴) and were cultured for 2 weeks to allow virus spread before the harvest of cells and analysis of FeLV proviral DNA by Southern blot hybridization. The detection of an internal 3.7-kb genome fragment provides a quantitative estimate of FeLV DNA content, as shown in Fig. 1C. Three distinct patterns were noted. HEK293 cells are permissive, and FeLV-B spread rapidly to saturation after infection at a low MOI. At the other extreme, the nonpermissive B-cell leukemia cell line Reh showed detectable proviral DNA only at an MOI of 1. The myeloid leukemia cell line KYO-1 showed an intermediate, partially permissive pattern, with the copy number diminishing in proportion to the infectious challenge dose. Quantitative PCR analysis of DNA from cells infected at an MOI of 1 indicated 30-fold-lower levels of FeLV DNA in Reh cells (∼0.3 copy/cell) than in semipermissive KYO-1 cells, which displayed levels similar to those in permissive controls (∼10 copies/cell).

As shown in Table 1, the infection/spread assay revealed full permissiveness (phenotype 1) for the replication of FeLV-B in 7 of 11 human cancer cell lines selected from the NCI-60 cancer cell panel (https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm ). Smaller numbers of cancer cell lines were partially permissive (phenotype 2) or nonpermissive (phenotype 3), with no obvious relationship to the level of expression of receptor PIT-1 mRNA as measured by quantitative reverse transcription-PCR (qRT-PCR), which was in almost all cases higher than that for the permissive HEK293 cell control) (Fig. 1D).

While spreading phenotype 1 was observed in multiple cancer cell lines, we noted that Hs68 primary foreskin fibroblasts were more restrictive. To explore the possibility that phenotype 1 is a feature of long-established cancer cell lines, we extended our study to a panel of recently established pancreatic cancer cell lines (34) as well as other nontransformed cells, including nonimmortalized CD34⁺ cord blood cells, lung fibroblasts (IMR90), and immortalized but nontransformed skin keratinocytes (HaCAT). Where cell numbers were limiting (e.g., for CD34⁺ cells), FeLV DNA content was analyzed by quantitative PCR. Pancreatic cancer cell lines displayed either the fully or the partially permissive phenotype, while the partially permissive phenotype was also observed in CD34⁺ cord blood cells. The fully permissive phenotype was observed in HaCAT and IMR90 cells, indicating that permissiveness is not simply a consequence of oncogenic transformation. It is also noteworthy that we observed no evidence of cytopathic effect or significant growth alteration in any of the FeLV-B-infected cell cultures, indicating that restricted viral spread was not due to loss of cell viability or interference with cell division. In contrast, FeLV-C variants have marked cytopathic effects in some cells (35).

The permissive spreading phenotype in multiple cancer cell lines is associated with the release of high-titer FeLV at levels comparable to those in feline fibroblast controls. To explore the basis of the restricted replication phenotypes in greater depth, we examined the expression of intracellular viral antigen and virion production in representative cell lines (KYO-1 and Reh) that were exposed to FeLV-B at MOI of >1 and cultured for 2 weeks to allow viral spread. To explore the hypothesis that hematopoietic cells are more widely resistant than other cell types, we extended this analysis to a wider panel of hematopoietic cell lines as well as primary human peripheral blood mononuclear cells (PBMCs). As shown in Fig. 2A, infected KYO-1 cells express the FeLV precursor polyprotein Pr65^Gag at levels similar to those in permissive feline fibroblast cells (AH927), while Reh cells and PBMCs produce low or undetectable levels. A similar divergence was noted when virion production was analyzed. At 14 days postinfection, partially permissive KYO-1 cells showed sustained release of virions at levels similar to those in permissive feline cells, while virion release by Reh cells and PBMCs was low to undetectable (Fig. 2A). In a separate experiment, the virtual equivalence of virion protein release was illustrated by serial dilution of pelleted virion preparations prior to Western blot analysis (Fig. 2B). As shown in Fig. 2C and Table 2, extending this analysis to a wider panel of hematopoietic cell lines showed that 9 of 13 leukemia cell lines displayed a KYO-1-like phenotype with sustained high-level release of virions, while the other 4 resembled Reh cells, with very low to undetectable levels of virus release. The level of permissiveness showed no clear pattern with regard to the type of leukemia (B cell, T cell, myeloid, erythroleukemia) or the cytogenetic features of the leukemia cell lines. PBMCs from four separate donors and a series of four Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs) showed a low to nonpermissive phenotype in this assay. An assay for infectious FeLV released by these cells revealed a similar pattern. Not surprisingly, cells with low virion release did not release significant levels of infectious FeLV. However, virus released at high levels from partially permissive lines showed very marked differences in specific infectivity, which was 4- to 10⁴-fold lower than that of virus released from permissive feline cells. The CEM cell line and its derivative CEMSS (36) were the most permissive lines tested, while Raji and LAMA84 cells released the least infectious virions (Table 2).

To explore the basis of the nonpermissive phenotype in greater detail, we examined the accumulation of proviral DNA after the exposure of cells to FeLV-B at an MOI of 1. Notably, nonpermissive PBMCs accumulated levels of FeLV DNA similar to those observed in partially permissive, high-virion-release KYO-1 cells (Fig. 3A). This was not due to detection of preexisting DNA in virions, since only very low levels were detected after the 30-min adsorption period, with significant increases by 6 h. To confirm that this accumulation is a receptor-dependent process, we compared FeLV-A infection of permissive feline lymphoma cells (3201 cells) to that of Reh cells, which do not express detectable mRNA for the human receptor homologue THTR1. This control was chosen because no PIT-1-negative cell lines were identified. The lack of accumulation of proviral DNA in the receptor-negative cells supports the hypothesis that this process is the result of receptor-mediated entry. The efficient detection of newly synthesized DNA with either long terminal repeat (LTR) or envelope gene primers suggests that complete proviral sequences are present, with no bias toward “strong-stop” products (37).

More-extensive analysis of postadsorption amplification of proviral DNA by quantitative PCR showed that all cells tested were initially permissive regardless of the later outcome of infection (Fig. 3B). However, comparison of the quantitative change in DNA from the 6-h time point to late infection (14 days) revealed a stark contrast between the semipermissive, high-virion-release cells (e.g., CEM, Raji, and Jurkat cells), where copy numbers increased over time, and the low-virion-output, nonpermissive cells (e.g., PBMCs and Reh cells), which showed unchanged or decreasing levels of proviral DNA. Intriguingly, EBV-immortalized cell lines mainly fell into the partially permissive category in this assay, suggesting that significant viral spread had occurred in these cells, while day-14 analysis revealed only low levels of virion release with no detectable infectivity. Again, we noted no cytopathic effect or changes in cell growth in these experiments.

Despite the lack of proviral DNA amplification, we noted that FeLV-B DNA was readily detectable in PBMCs at similar levels between 3 and 14 days after initial infection (Fig. 4A). This stable association suggested that FeLV-B DNA may be integrated into PBMCs, and this hypothesis was tested further by quantitative PCR after preamplification with a FeLV Gag primer and a consensus human Alu repeat sequence (Fig. 4B). The greatly enhanced detection of FeLV DNA after Alu-Gag preamplification indicates that much of the DNA is in an integrated form. The possibility that FeLV DNA persists in an unintegrated form was also tested by PCR for circular forms generated by ligation in the nucleus. LTR circles were barely detectable in PBMCs and Reh cells by 3 days postinfection. Positive controls for this assay were early (24-h) samples from productively infected 3201 and HEK293 cells, where single and double LTR circles were detected (Fig. 4C). Cloning and sequencing confirmed the identity of the single-LTR form (not shown).

To explore the hypothesis that APOBEC cytidine deaminase activity is involved in FeLV growth restriction, we analyzed proviral DNA after PCR amplification of genome fragments from infected cells. These sequences were compared to the index FeLV-B (pFGB) sequence for evidence of characteristic G→A mutations that are induced during reverse transcription (38). We found evidence of extensive hypermutation of FeLV replicating in human cells at rates as high as 7 mutations/kb. This mutation rate is much higher than the G→A mutation rate reported for FeLV replicating in cats in vivo (0.1/kb) (39), indicating that FeLV, like MLV (30), is able to evade this restriction factor only in its natural host. FeLV strains typically encode a glycosylated Gag precursor open reading frame (40), and this is intact in the Gardner-Arnstein FeLV-B molecular clone. However, FeLV, like MLV, is sensitive to heterologous human APOBEC3 activity (41). As shown in Fig. 5 with the examples of the Burkitt lymphoma cell line Raji and the pancreatic cancer cell line TKCC15LO, mutational footprints indicated that the predominant signature was GG→GA, suggesting that APOBEC3G (A3G) is responsible (42). Moreover, expression of A3G mRNA showed a positive correlation with mutational activity when all cell lines were compared (R = 0.22; P = 0.01). The results are collated in Fig. 5B and are broken down according to the permissive (phenotype 1), partially permissive (phenotype 2) (high virion release), or nonpermissive (phenotype 3) (low virion release) phenotype. Not surprisingly, permissive cells showed low A3G expression and low mutational activity, while partially permissive, high-virion-release cells showed high A3G expression and mutational activity. In contrast, nonpermissive cells showed no evidence of hypermutation, indicating that a nonmutational mechanism of resistance is operative in these cells. Moreover, a lack of mutation in PBMCs was observed despite very high levels of A3G, suggesting that the proviral DNA detected was the result of initial reverse transcription rather than secondary spread. The four LCLs showed significant levels of hypermutation (Fig. 5B, center, dashed oval), supporting their grouping with the partially permissive cells on the basis of proviral DNA accumulation (Fig. 3).

Analysis of all G→A mutations in the cell lines with significant levels of hypermutation (Fig. 5C) indicated that A3G is the major, though not the sole, effector of G→A mutations in the FeLV genome during replication in human cells. Notably, the two cell lines with divergent signatures (Hs68 and KYO-1) displayed high levels of APOBEC3F mRNA (not shown). The expression levels of A3G mRNA detected ranged over several log units but were notably higher in cells of hematopoietic origin (Fig. 5D). The CEM cell line and it derivative CEMSS showed the lowest levels among hematopoietic cell lines, in agreement with their low mutation rates and high levels of release of relatively noninfectious FeLV virions (Table 2). High levels of A3G mRNA relative to that in the HEK293 control were also associated with restricted FeLV spread in nonhematopoietic cells, but the majority of these lines showed low expression and a fully permissive phenotype. However, as indicated in Fig. 5D, the nonpermissive phenotype (nonspreading phenotype 3 [Table 1] and low proviral DNA accumulation [Fig. 3]) shows no relationship with A3G expression or the mutation rate. This finding, taken together with evidence that this phenotype arises from a postintegration block to incoming virus, suggests that this is an A3G-independent barrier.

FeLV-B stocks were generated by transfection of an infectious molecular clone (pFGB) (55) and propagation in the feline fibroblast cell line AH927 or HEK human embryonic kidney cells as described previously (56). For FeLV-A stocks, HEK293 cells were transfected with pFGA-5 (20) to avoid possible recombination and generation of FeLV-B (57). FeLV infectivity was determined by titration on S⁺ L⁻ QN10 cells (33).

Cell lines were derived from curated frozen stocks maintained in our laboratory (Jurkat, K562, CEM, Reh, Kasumi, U937, MCF-7) or by colleagues in the Glasgow Cancer Research UK Centre, including A. Biankin (pancreatic cancer TKCC series) and T. Holyoake (chronic myeloid leukemia lines KYO-1, BV173, KU812, LAMA84, and EM-2; B-cell acute lymphoblastic leukemia lines ALL/MIK and TOM-1). IMR90 fibroblasts were provided by P. Adams (48), while EBV-transformed lymphoblastoid cell lines (LCLs) were obtained from R. Jarrett, and HaCAT keratinocytes were provided by S. Graham. CEMSS cells were kindly provided by M. Malim (King's College, London, United Kingdom). CD34⁺ cells were purified by positive selection using anti-CD34-conjugated magnetic microbeads (catalog no. 130-046-703; Miltenyi Biotec Inc.) from cord blood samples generously provided by the Children's Cancer and Leukemia Group (CCLG). Primary blood mononuclear cells were obtained from Cambridge Bioscience (1 sample) and the Scottish National Blood Transfusion Service (3 samples).

A total of 4 × 10⁵ KYO-1 cells or Reh cells or 5 × 10⁴ HEK293 cells were plated per well in 6 wells of a 12-well plate the day before infection. FeLV-B was harvested from subconfluent virus-producing HEK293 cells, filtered through 0.45-μm filters, and serially diluted 1:10 in RPMI 1640 medium with 10% fetal calf serum (FCS). A 0.5-ml volume of filtered diluted virus was added per well, or medium alone was added to the control well, for 2 h. Cells were then fed with fresh medium and were expanded as normal for 14 days. Genomic DNA was extracted from phosphate-buffered saline (PBS)-washed, pelleted cells using a Qiagen DNeasy blood and tissue kit. Aliquots (15 μg) of genomic DNA were digested overnight with KpnI, separated on a 0.8% agarose Tris-acetate-EDTA (TAE) gel, transferred to a Hybond N membrane (Amersham) in 20 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and hybridized to FeLV ExU3 as described previously (21).

RNA was extracted from cultured cells using a Qiagen RNeasy minikit, and cDNA was synthesized from 1 μg RNA using a Qiagen QuantiTect reverse transcription kit. Aliquots (12.5 ng) of cDNA were amplified in triplicate using Power SYBR green PCR master mix (Thermo Fisher Scientific) and Qiagen QuantiTect primer assays for human hypoxanthine phosphoribosyltransferase (HPRT) (QT00059066), APOBEC3G (QT00070770), SLC20A1 (PIT-1) (QT00028763), or SLC19A2 (THTR1) (QT00007847) on an ABI 7500 real-time PCR system. Quantification was carried out relative to HEK293 cells, and values were normalized to HPRT expression.

One hundred-nanogram aliquots of genomic DNA were amplified in Reddy Mix (Thermo Fisher Scientific) using primers FeLV envA F (5′-TAAAACACGGGGCACGTTAC-3′) and FeLV envA R (5′-GGAGGTGGGCTTCCACCAAG-3′), FeLV envB F (5′-ATGTGATCAGCCTATGAGGA-3′) and FeLV envB R (5′-CACTAGCTCCCGTTGTCGAG-3′), FeLV LTR F (5′-TAGCTGAAA CAGCAGAAGTTTCAAG-3′) and FeLV LTR F R (5′-GGAAGGTCGAACTCTGGTCAAC-3′), and Myc F (5′-CCAACAGGAACTATGACCTCG-3′) and Myc R (5′-GTAGAAGTTCTCCTCCTCGTC-3′). Samples were denatured at 94°C for 3 min and were then subjected to 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Amplification products were separated by electrophoresis on 2% agarose-TAE gels and were visualized by ethidium bromide staining.

Genomic DNA was extracted by a Qiagen DNeasy minikit, and 20-ng aliquots were amplified using primers for human β2 microglobulin (F, 5′-GGAATTGATTTGGGAGAGCAT-3′; R, 5′-CAGGTCCTGGCTCTACAATTTACTAA-3′), primers FeLV U3-LTR F (5′-TAGCTGAAA CAGCAGAAGTTTCAAG-3′) and FeLV U3-LTR R (5′-GGAAGGTCGAACTCTGGTCAAC-3′), and primers envB F (5′CGGTATCCCGGCAAGTAATG-3′) and envB R (5′-GGTTTTTGATCAGGCAGGACTAGA-3′) in Power SYBR green PCR master mix. Amplification products were detected on an ABI 7500 real-time PCR system. Samples were normalized to β2 microglobulin.

PBS-washed, pelleted cells were lysed in whole-cell lysis buffer and protein concentrations measured by a Bio-Rad protein assay. Virion lysates were prepared from 2 ml precleared virion supernatant by centrifugation in a Beckman TL100 benchtop ultracentrifuge for 2 h at 37,000 rpm and 4°C. Virion pellets were resuspended in 75 μl lysis buffer, and an equal volume (13 μl) of virion protein or 10-μg aliquots of cellular protein were separated on 4-to-12% NuPAGE Novex Bis-Tris gels in NuPAGE morpholinepropanesulfonic acid (MOPS) SDS buffer and were transferred to Hybond ECL membranes (Amersham) in NuPAGE transfer buffer. Following overnight blocking in 5% milk in Tris-buffered saline–Tween 20 (TBST), the filter was exposed to a 1:500 dilution of an anti-p27^Gag monoclonal antibody (VPG19.1; a gift of Brian Willett), and detection was carried out with the ECL detection reagent (GE Healthcare).

FeLV gag or pol fragments were generated by amplification of 100-ng aliquots of infected-cell genomic DNA in Reddy Mix PCR master mix (Thermo Fisher) using primers FeLV p27gag F (5′-CCCAGTGGCCCTAACTAACC-3′) and R (5′-GCTGGCGTTTCCTCTTTCC-3′) or FeLV Pol F (5′-GCAACCGGTAAGGTGACTC-3′) and Pol R (5′-TTCAAAGGGTTTGGTGATATCTG-3′). DNA was denatured at 94°C for 3 min and was then put through 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Products were purified through Qiagen QIAquick PCR purification columns and were then cloned into pCR2.1-TOPO (Invitrogen), transformed into TOP10 competent cells, and plated onto Amp X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) agar plates. White colonies were cultured and plasmid DNA extracted using Qiaprep spin preps, and DNA was sequenced from M13 F and M13 R primers by Source BioScience and was examined for mutations relative to the pFGB molecular clone using Hypermut, version 2.0.

Primary PCR on 50 ng genomic DNA was performed in Platinum Taq DNA polymerase (Invitrogen) using 10 μM primers AluF (5′-GCCTCCCAAAGTGCTGGGATTACAG-3′) and FeLV gag R (5′-ATAGGGAGGTGGCTCTTCTG-3′) at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 50°C for 15 s, and 72°C for 3 min 30 s. Nested PCR was carried out on 4 μl primary PCR product or 4 ng unamplified DNA using primers FeLV RU5 F (5′-TCTTTGCTGAGACTTGACCG-3′) and FeLV RU5 R (5′-ACTAGGTCTTCCTCGGCGAT-3′) and probe FeLV RU5 (5′-FAM-TGCATCTGACTCGTGGTCTC-TAMRA-3′) using Takyon LowRox Probe Mastermix dTTP blue (UF-LPMT-BO101) (Eurogentec) and settings of 50°C for 2 min and 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 60 s on an ABI 7500 real-time PCR system.

Aliquots (100 ng) of genomic DNA from 3-day (PBMCs, Reh and KYO-1 cells) or 24-h (3201 and HEK293 cells) FeLV-infected cells were amplified in Reddy Mix PCR master mix with primers Circles env F (5′ TAAAACAGCGGCAACAACTG-3′) and Circles gag R (5′-GTCTCCGATCAACACCCTGT-3′) or the positive-control primers pan-Myc 23F (5′-CCAACAGGAACTATGACCTCG-3′) and pan-Myc 96R (5′-GTAGAAGTTCTCCTCCTCGTC-3′). Following an initial denaturation step at 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min 30 s were performed. Portions (15 μl) of each 25-μl reaction mixture were separated on 1.4% agarose-TAE gels and were visualized by staining with ethidium bromide.