- Open Access
Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms
© Nitta et al. 2015
- Received: 17 February 2015
- Accepted: 23 July 2015
- Published: 8 August 2015
Koala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag.
In this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results.
These results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.
Koala retrovirus (KoRV) is a recently discovered retrovirus that infects koalas , and it is a likely candidate for the causative agent of lymphomas and other hematologic diseases in these animals. There are several interesting and unique features of KoRV. First, some but not all koalas carry endogenous KoRV-related proviruses, and among different unrelated animals the patterns of endogenous proviral integration sites are distinct . It has also been reported that endogenization of KoRV into koalas is an ongoing process . Thus it appears that KoRV has infected koalas recently, to the extent that individual endogenous KoRV proviruses have not become fixed into the koala population. This contrasts with the situation for endogenous retroviruses in many other species, including humans, where most individual proviruses are shared by all members of the species. Second, KoRV infection appears to be spreading from north to south in Australia, with virtually all animals in the north showing evidence for infection (both endogenous and potentially exogenous KoRVs); in southern regions and on some offshore islands, the incidences of KoRV infection and/or endogenization are lower .
KoRV is a member of the gammaretrovirus genus. Gammaretroviruses encode the standard three genes of retroviruses, gag, pol and env, but no accessory proteins such as those of lentiviruses, deltaretroviruses, epsilonretroviruses and betaretroviruses. KoRV’s closest relative is gibbon-ape leukemia virus (GaLV), and it is more distantly related to the murine leukemia viruses (MuLVs) . The predominant KoRV (endogenous and exogenous) is now referred to as KoRV-A, that infects cells by binding to the Pit-1 phosphate transporter as a receptor . Other KoRV isolates (e.g. KoRV-B and others) differ from KoRV-A in the envelope gene , and they infect cells via different receptors (e.g. the thiamine transporter for KoRV-B) . It has been suggested that KoRV-B infection is associated with development of lymphoma . KoRV may result from relatively recent cross-species infection of an MuLV-like virus of a southeast Asian rodent into koalas.
We and others have shown that many gammaretroviruses encode an alternate form of Gag polyprotein, glycosylated Gag or glyco-gag [8–10]. We have shown that glyco-gag of Moloney MuLV enhances virus replication by facilitating virus release through lipid rafts  and antagonizing a host restriction factor, mouse APOBEC3 (mA3) . Glyco-gags in MuLVs are translated from unspliced viral RNA from an upstream CUG initiation codon in the same reading frame as the AUG codon used for initiation of Gag and Gag-Pol polyproteins. However some endogenous MuLVs (of the class A xenotropic, polytropic and modified polytropic classes) do not have the capacity to encode glyco-gag . The KoRV genome contains an in-frame ORF upstream of Gag characteristic of glyco-gags of exogenous and endogenous MuLVs . In this report, we tested if KoRV expresses a glyco-gag, and if glyco-gag is important for infectivity in vitro. In light of the role of glyco-gag in counteracting restriction by APOBEC3 for M-MuLV we also investigated restriction of wild-type and a putative glyco-gag negative version of KoRV by murine and human APOBEC3s.
Use of DERSE cells to detect KoRV infection
Absence of glycosylated gag expression in human cells infected by KoRV
Restriction of KoRV infection by APOBEC3 proteins
The effects of APOBECs on KoRV infection were also examined in cells stably infected with KoRV by transfecting plasmids expressing APOBEC3s into 293T/gg- and 293T/WT cells and measuring the infectivity of virus released from these cells by the DERSE cell assay. Transfection of hA3G and mA3∆E5 into both of these cells showed ca. 40–50% reduction in KoRV infectivity (not shown). These effects were less pronounced than those observed in Fig. 6, where transient co-transfections of KoRV and APOBEC3 expression plasmids were carried out. This may have been due to the fact that the efficiencies of transfection in the 293T/gg- and 293T/WT cells were 50–70%; thus 30–50% of these infected cells would not have expressed the corresponding APOBEC3s. In contrast, in plasmid co-transfections, cells taking up DNA tend to receive and express both plasmids .
hA3G but not mA3∆E5 induces G-to-A hypermutation in KoRV infection
Clones (No. of mutants)/No. of clones sequenced
No. of bases sequenced
No. of mutations
No. G-to-A mutations (frequency)
No. of bases (bp)/G-to-A mutation
Local sequence +1 (A/T/C/G)
Local sequence −1 (A/T/C/G)
One potential concern with the results shown in Table 1 could be if multiple PCR products from an individual reverse transcribed viral DNA were cloned and sequenced, which could have led to distortion of the calculated mutation frequencies and over-estimates of the number of bases sequenced. However, for the analysis of KoRV/hA3G DNA, of 23 clones sequenced showing mutations, the mutation patterns for 22 of them were distinct—two clones showed the same pattern and were considered to be from the same original viral DNA. Therefore the data in Table 1 largely represent sequences from different viral DNAs.
In this study, we studied two aspects of KoRV in the context of infectious virus. We first developed an assay system that could conveniently assess KoRV infectivity, through the use of DERSE cells. We then tested if KoRV encodes glyco-gag protein and the results indicated that even though KoRV appears to have the capacity to encode a glyco-gag, it does not do so, at least in human cells. We also tested if APOBEC3 restriction factors can restrict KoRV infection, and found that both hA3G and mA3∆E5 potently restrict KoRV to similar degrees. However while hA3G restriction was accompanied by extensive cytidine deamination, mA3∆E5 restriction was not. Thus restriction of KoRV by these two proteins occurs by predominantly different mechanisms.
DERSE cells were developed as an assay system for infectious retroviruses based on their ability to co-package an XMRV-based RNA containing GFP sequences and transfer it to other DERSE cells by retroviral infection. The fact that KoRV infection of DERSE cells resulted in spread of green fluorescence indicated that KoRV Gag polyprotein can incorporate XMRV RNA into virions, which further suggested that KoRV Gag can recognize the XMRV RNA packaging (Psi) sequence. Indeed there was also extensive spread of both GFP and KoRV Gag to 293T cells infected with the KoRV released from the infected DERSE cells (Fig. 1). Rapid assessments of infectious KoRV can be conducted by western blotting for KoRV Gag or GFP (Fig. 2) or flow cytometry for GFP; highly sensitive quantification could be accomplished by focal immunofluorescence assays. How XMRV RNA interacts with KoRV Gag and/or nucleocapsid protein remains to be determined, but XMRV and KoRV share 59.8% total nucleotide sequence identity (aligned by the Clustal W method, DNASTAR’s MegAlign), so such an interaction is plausible. Likewise alignment of the KoRV sequence with the regions of M-MuLV and XMRV RNAs predicted by mutational and SHAPE analysis  to contain their Psi sequences suggests a putative KoRV Psi sequence from nt 565–753 (T. Nitta & H. Fan unpublished). M-Fold predicts a possible stem-loop structure in the center of this sequence, which could be consistent with its function as the KoRV Psi.
One unique feature of gammaretroviruses is that many encode a glyco-gag protein initiated from an in-frame CUG codon upstream of AUG start codon for Gag polyprotein . We and others have shown that MuLV glyco-gag facilitates virus release/assembly [11, 13, 30], protects the reverse transcription complex in viral cores from restriction by mA3 , it rescues infectivity of Nef-deficient HIV-1 [31, 32] and it decreases HIV-1 sensitivity to neutralizing antibodies . Phylogenetic comparisons of gammaretroviruses of different species reveal equivalent putative glyco-gag open reading frames with the conserved N-terminal LGDVP motif for viruses ranging from KoRV and Gibbon-Ape leukemia virus (GaLV) through exogenous MuLVs and endogenous gammaretroviruses of mice (XMV class B and C) and humans (HERV-H) . The fact that the glyco-gag open reading frame has been conserved among exogenous retroviruses such as KoRV and GaLV strongly suggests that this protein is expressed and biologically important; its broad distribution among exogenous and endogenous gammaretroviruses suggests that it is an ancient function of these viruses. In this light, it was surprising that the experiments in this study did not detect evidence for expression of glyco-gag by KoRV: (1) neither 293T cells nor DERSE cells productively infected with wild-type KoRV showed glycosylated forms of Gag, (2) mutation of the putative CUG initiation codon for KoRV glyco-gag did not affect virus release efficiency or infectivity, and (3) restriction by both hA3G and mA3∆E5 was efficient and equivalent for WT and KoRV gg-. The pKoRV gg- plasmid contained a stop codon within the LGDVP motif in the putative KoRV glyco-gag coding sequences so it would not encode the most likely form of glyco-gag. In future experiments, it will be interesting to determine the molecular basis for why KoRV does not encode a glyco-gag even though it has the putative coding sequences including three CUG codons.
The apparent lack of glyco-gag expression by KoRV in these experiments might suggest that this protein is dispensable for in vivo infectivity, but on the other hand the conservation of the glyco-gag ORF in KoRV suggests that it is important biologically. One possible explanation could be that KoRV glyco-gag is expressed in cells of its natural host the koala, but not in human cells, which will be interesting to test. Alternatively the maintenance of the putative glyco-gag open reading frame in KoRV could potentially reflect the fact that KoRV glyco-gag is not required for KoRV infection although it was descended from gammaretroviruses (e.g. the GaLV/KoRV progenitor) that do/did express and depend on this protein. KoRV may have been infecting koalas for a relatively short time , which may not have been long enough for glyco-gag mutations to have arisen and become fixed in the KoRV genome.
The relationship of glyco-gag and resistance to APOBEC3 restriction for gammaretroviruses is of interest. As described above, glyco-gag is important for resistance of M- and F-MuLV to mA3 in vivo and in vitro [12, 13, 19]. On the other hand, some endogenous MuLVs (XMV class A and the polytropic and modified polytropic PMVs and MPMVs) do not have the capacity to encode a glyco-gag, and sequence analysis suggests that they lost the function . It is striking that endogenous PMV and MPMV proviruses show evidence for G → A mutations, while endogenous XMVs (including class B and Cs that are glyco-gag positive) do not . In fact the replication competent XMRV (derived by recombination between two XMVs) does not encode functional glyco-gag . Interestingly replication of this virus can be potently inhibited by both human and murine APOBEC3s, accompanied by G → A hypermutation . In contrast exogenous M-MuLV is actually relatively resistant to mA3 compared to hA3G; G → A hypermutation is not observed for mA3, but it is for hA3G . This might suggest that glyco-gag is responsible for the differential responses to mA3 and hA3G. In this light the restriction of KoRV infection by mA3 and hA3G was interesting. Both of these factors strongly and equivalently inhibited wt KoRV infection in 293T and DERSE cells, where glyco-gag protein was not detectable. However hA3G restriction was accompanied by extensive G → A hypermutation, while mA3 restriction showed substantially less deamination and hypermutation [although at levels above background (no mA3)]. These results make several points: (1) for KoRV, the mechanisms of restriction are by and large different for hA3G (G → A hypermutation) and mA3 (little mutation), (2) glyco-gag is not determining the response to these factors for KoRV, and (3) some other function or protein of KoRV is responsible for the differential response to mA3 and hA3G. It seems possible that these results may reflect the fact that KoRV was descended from a murine gammaretrovirus, and the murine gammaretroviurses have evolved to replicate in the presence of mA3.
It is noteworthy that mA3 restriction of Moloney and Friend MuLV is accompanied by virtually no G → A mutations, while restriction of Akv MuLV does show measurable mutations . It has recently been reported that this difference maps to the respective glyco-gags of these viruses, and in particular to their levels of glycosylation . It will be interesting to compare the low level G → A mutation observed in mA3 restriction of glyco-gag negative KoRV to that of Akv MuLV.
It is also interesting that marsupials have been reported to lack APOBEC3 genes . The lack of an APOBEC3 gene in koalas would be consistent with the absence of glyco-gag in KoRV, if resistance to APOBEC3 proteins has been a driving force in maintaining glyco-gag during gammaretroviral evolution. As noted in the previous paragraph, the mechanisms of KoRV restriction by hA3G and mA3 may reflect evolution of KoRV from murine gammaretroviruses.
In these experiments, co-expression of mA3 (and hA3B, not shown) decreased the levels of KoRV Gag protein in cells, in addition to inhibiting viral infectivity. The reduction in Gag protein was specific for KoRV, since parallel experiments with M-MuLV did not show this effect. The mechanism(s) of the Gag inhibition remain to be determined, and will be the subject of future experiments. Conceivably this is another mechanism of restriction for these antiviral proteins.
The fact that KoRV can productively infect human cell lines has been described previously and was confirmed here for 293T and DERSE cells. This might suggest concerns for zoonotic infection of humans, particularly in light of the suggestions of KoRV-associated pathologies and malignancies in koalas. However, there have been no reports of transmission of KoRV to humans with significant occupational exposure, e.g. veterinarians or animal keepers . The strong restriction of KoRV by hA3G described here provides support for the low likelihood of zoonotic transmission to humans; hA3G is highly expressed in hematopoietic cells, which are prominent targets for gammaretroviral infection. The 293T and LNCaP cells (from which DERSE cells were derived) that support KoRV replication are known to have little or no hA3G activity . Similar considerations contributed to the conclusions that XMRV is not a human pathogen .
A convenient assay for assessing KoRV infectivity in vitro was employed with DERSE cells. No evidence for expression of a functional glyco-gag in KoRV was observed. KoRV replication was restricted by both mA3 and hA3G regardless of the mutation in the putative glyco-gag sequence. hA3G restriction induced extensive G → A hypermutation during reverse transcription while mA3 restriction did not, suggesting that human and mouse APOBEC3s inhibit KoRV replication in different manners.
Cells and DNA constructs
HEK293T cells  were grown in Dulbecco’s modified Eagle’s medium supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum. DERSE.LiGP (DERSE, Detectors of Exogenous Retroviral Sequence Elements) cells (kindly provided by Dr. Vineet Kewalramani  were grown in RPMI1640 supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), G418 (100 μg/ml) and 10% fetal bovine serum. The full-length molecular clones of KoRV, pKoRV522  and of M-MuLV, p63-2  were described previously. The KoRV mutant, pKoRV gg- that contains a stop codon in the putative KoRV glyco-gag coding sequences (TAG at nt 776–778—NCBI Accession number AB721500) was made by site-directed mutagenesis according to standard techniques . The viral sequence was amplified from pKoRV522 with PfuUltra II Fusion HS DNA Polymerase (Agilent Technology) and the oligomers GACCAGGGACGCCTGTAGGACCCCACGGCAAG and CTTGCCGTGGGGTCCTACAGGCGTCCCTGGTC. The PCR products were digested with DpnI and then transformed with DH5α. The introduced mutation was confirmed by sequencing. The plasmids expressing, V5-tagged hA3G  and FLAG-tagged mA3∆E5  were described previously. The plasmids pcDNA3.1(+) and pEGFP-N1 were obtained from Invitrogen and used as controls.
Antibodies and Chemicals
Rabbit polyclonal anti-MuLVp30CA antiserum  and anti-KoRV CA  antiserum were described previously. For detection of epitope tags, mouse and rabbit anti-Flag antibodies (Cell Signaling), anti-V5 (Invitrogen) and anti-GFP (Biovision) were used. Beta-Tubulin levels were used to assess sample loading in the gels and were detected by rabbit anti-beta-Tubulin (Cell Signaling). For western blots, we used anti-mouse IgG conjugated with horseradish peroxidase (Thermo Scientific) and anti-rabbit IgG conjugated with horseradish peroxidase (GE Healthcare).
Detection of viruses and assessment of viral release efficiency
293T and DERSE cells stably infected with wild-type and mutant KoRVs were established by infection with KoRVs produced from 293T cells transiently transfected with pKoRV522 and pKoRV gg-. Detection of infection in the infected cells was by immunofluorescent microscopy for GFP and western blots for KoRV CA protein. The DERSE cells infected with KoRV showed bright GFP fluorescence 6 days post-infection and the fluorescence images were taken with the Axiovert200 microscope (Carl Zeiss). Similarly, GFP in the 293T cells infected with KoRV released from the stably infected DERSE cells was detectable by fluorescence microscopy. For these experiments, the cells were gathered 6–7 days post-infection for detecting KoRV CA protein by western blots. Western blots and assessment of virus release were performed as described previously  with slight modifications. In brief, the KoRV-infected 293T and DERSE cells were seeded on 6-cm dishes 1 day before measuring viral release. The media were replaced once and cells were incubated for further 6 h after which both cells and media were gathered. The media were clarified by low-speed centrifugation (10 min at 2,000g), passed through a 0.45 μm filter and viral particles were pelleted in a Beckman SW41 rotor at 77,000g for 1 h. The pelleted viral particles and corresponding cell lysates were analyzed by SDS-PAGE and western blots using anti-KoRV CA and chemiluminescent detection with an AlphaImager system (Alpha Innotech). To quantify viral release, Pr60 gag , Pr50 gag and CA bands in the cells and media were quantified with the densitometry software AlphaEaseFC (Alpha Innotech), and the percentage of released Gag divided by total Gag proteins (Pr60 gag , Pr50 gag and CA) in cells and media was calculated. Different exposures of the blots were analyzed to ensure that densitometry was in the linear range.
Assessment of antiviral effects of human and mouse APOBEC3s
293T cells were co-transfected with the plasmids expressing KoRVs (pKoRV522 or pKoRV gg-) and APOBEC3s or control plasmids using the CalPhos Mammalian Transfection Kit (Clontech Laboratories). Similarly 293T cells stably infected with KoRVs (293T/WT and 293T/gg-) were transfected with the same APOBEC3 expression plasmids. The media were replaced 6–8 h after transfection and the cells were incubated further for 40 h, after which both the cells and media were harvested. In the experiments using 293T cells stably infected with KoRV, media were replaced again 48 h after APOBEC3 plasmid transfection and the both cells and media were collected 24 h after the second media replacement. Equal portions of the viral and cellular samples were subjected to SDS-PAGE and western blotting for KoRV Gag and APOBEC3s. To evaluate antiviral effects of human and mouse APOBEC3s on KoRV infectivity, DERSE cells were infected with KoRVs released from 293T cells expressing both KoRV and APOBEC3s. A total of 105 DERSE cells were seeded on 6-cm dishes 1 day before infection and the infected cells were gathered 6–7 days post-infection. The cells were lysed by the lysis buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% (vol/vol) Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 20 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Complete EDTA Free, Roche Diagnostics) and the same volumes of whole cell lysates were subjected to SDS-PAGE and western blots using anti-KoRV CA antibodies. The blots were analyzed by densitometry as described above. The results were normalized for the amounts of the viruses used for infection.
Assessment of mutations induced by human and mouse APOBECs in KoRV infection
DERSE cells were infected with KoRV prepared from the 293T cells co-transfected with pKoRV522 along with hA3G-V5, mA3∆E5-FLAG or pcDNA3.1. The infected DERSE cells were gathered 36 h post-infection, DNA was extracted, treated with DpnI and a portion of the KoRV pol region (NCBI AB721500, nt 5,637–6,668) was PCR amplified using PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies) and the primers 5′-TGCGTCTGGGGAAGTCGTGGG and 5′-CTACTACCGGTGGGGGACTTG. The PCR products were cloned using the Zero Blunt® TOPO® PCR Cloning Kit (Life Technologies) and sequences from individual clones were compared to the original KoRV sequence. PCR products from three or more amplifications were used in two independent infection experiments; the resulting PCR products were cloned into the vector pCR™4Blunt-TOPO® Vector and multiple clones were subjected to sequencing by a commercial vendor (Eton Biosciences).
TN designed and performed experiments, interpreted the data and drafted the manuscript. DH and FG performed experiments and provided helpful discussion regarding data analysis. TM provided KoRV plasmids, antibodies against KoRV and discussion for the manuscript. HF supervised the project, designed the research, interpreted the data and prepared the manuscript. All authors read and approved the final manuscript.
We thank Tom Hsu, David Camerini, Yoko Nitta, Jonathan Lambright, the UCI Cancer Research Institute, and the Optical Biology Shared Resource of the Chao Family Comprehensive Cancer Center for kind advice and technical assistance. We are grateful to Dr. Vineet Kewalramani for the gift of DERSE cells, and to Dr. Marc-Andre Langlois for FLAG-tagged mA3∆E5 plasmid. This work was supported by NIH Grant CA94188 to H. F. T.N. was supported in part by a JSPS Postdoctoral Fellow for Research Abroad.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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