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Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms.

Nitta T, Ha D, Galvez F, Miyazawa T, Fan H - Retrovirology (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, 92697-3905, USA. nittat@savannahstate.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Comparison of WT and putative glyco-gag-mutated KoRVs in viral production. a DERSE cells were infected with WT and glyco-gag-mutated (gg-) KoRVs produced from 293T cells transfected with pKoRV522 and pKoRV gg-. Gag in the cell lysates and media were detected by western blots using anti-KoRV CA antibodies. Western blotting for beta-Tubulin in the cell lysates confirmed equal loading of samples (not shown). b Cell lysates from 293T cells transfected with pKoRV522 or from the M-MuLV infected cell line 43D were treated with PNGase (endoglycosidase) F to remove N-linked oligosaccharides, and Gag proteins were detected by SDS-PAGE and western blots using anti-KoRV CA and anti-MuLV p30 antibodies. The locations of the Pr65gag Gag polyprotein precursor as well as a major cleavage product (Pr55gag) are shown, as well as the corresponding proteins (Pr60gag and Pr50gag) for KoRV. M-MuLV glyco-gag (the primary translation product gPr80gag as well as more slowly migrating forms with additional glycosylation) is indicated; endo F treatment reduced the size of gPr80gag to 75 kDa [48]. Virus release efficiencies of gg- KoRV compared to WT KoRV (set at 1 in each experiment) are shown for infected DERSE (c) and 293T (d) cells. The release efficiency measurements resulted from at least three independent experiments; error bars indicate standard deviation.
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Fig4: Comparison of WT and putative glyco-gag-mutated KoRVs in viral production. a DERSE cells were infected with WT and glyco-gag-mutated (gg-) KoRVs produced from 293T cells transfected with pKoRV522 and pKoRV gg-. Gag in the cell lysates and media were detected by western blots using anti-KoRV CA antibodies. Western blotting for beta-Tubulin in the cell lysates confirmed equal loading of samples (not shown). b Cell lysates from 293T cells transfected with pKoRV522 or from the M-MuLV infected cell line 43D were treated with PNGase (endoglycosidase) F to remove N-linked oligosaccharides, and Gag proteins were detected by SDS-PAGE and western blots using anti-KoRV CA and anti-MuLV p30 antibodies. The locations of the Pr65gag Gag polyprotein precursor as well as a major cleavage product (Pr55gag) are shown, as well as the corresponding proteins (Pr60gag and Pr50gag) for KoRV. M-MuLV glyco-gag (the primary translation product gPr80gag as well as more slowly migrating forms with additional glycosylation) is indicated; endo F treatment reduced the size of gPr80gag to 75 kDa [48]. Virus release efficiencies of gg- KoRV compared to WT KoRV (set at 1 in each experiment) are shown for infected DERSE (c) and 293T (d) cells. The release efficiency measurements resulted from at least three independent experiments; error bars indicate standard deviation.

Mentions: As mentioned in the introduction, the KoRV genome contains sequences that could potentially encode a typical glyco-gag. Examination of the KoRV (J group) RNA sequence indicated that KoRV has three potential upstream CUG codons in the same reading frame as the Gag polyprotein Pr60gag (Fig. 3). Moreover one protein sequence motif conserved in other gammaretroviral glyco-gags is present in the putative glyco-gag of KoRV: LGDVP at the N-terminus if initiation is at the CUG at nt 736. In addition a stretch of hydrophobic (potential membrane-spanning and/or signal peptide) amino acids is immediately upstream of the AUG for Pr60gag as for other gammaretroviral glyco-gags. There are three major KoRV isolates with different biological properties (KoRV-A, KoRV-B and KoRV-J) [18], and all of them showed nearly identical nucleic acid and protein sequences beginning with the conserved LGDVP motif in the leader peptide sequence (Additional file 1: Figs. S1, S2). To assess whether KoRV produces functional glyco-gag protein analogous to those in MuLVs, we introduced a mutation that would disrupt expression of putative glyco-gag protein in the plasmid containing the full-length KoRV molecular clone, pKoRV522 (Fig. 3); this plasmid was termed pKoRV gg-. WT and putative glyco-gag mutant KoRV stocks were prepared by transiently transfecting 293T cells with pKoRV522 and pKoRV gg-, and then used to infect DERSE or 293T cells. The infected cells were serially passaged until they all were infected, resulting in the stably infected cells DERSE/WT, DERSE/gg-, 293T/WT and 293T/gg-. As shown in Fig. 4a and quantified in Fig. 4c, the levels of Pr60gag in DERSE cells infected with WT and glyco-gag mutant KoRV were equivalent, as were the amounts of CA (virus) released into the media. Likewise 293T cells infected with the two viruses showed equivalent efficiencies of release (Fig. 4d). These results suggested that KoRV glyco-gag may not enhance virus release. On the other hand, western blots using anti-KoRV CA on the WT KoRV-infected cells did not show higher molecular weight proteins in addition to Pr60gag, which would possibly indicate that the infected DERSE and 293T cells did not express KoRV glyco-gag. In cells infected with WT MuLVs, glyco-gag proteins were sometimes somewhat diffuse in SDS-PAGE due to their glycosylation, and they migrated as more distinct and rapidly migrating bands if the glycosylation was removed [9]. As shown in Fig. 4b, treatment of cell extracts from WT M-MuLV infected cells with PNGase F (endoglycosidase F) showed a shift in mobility of the glyco-gag proteins (gPr80gagand gPr95gag-, right lanes). On the other hand, there was no evidence for equivalent proteins in the cells infected with WT KoRV (left lanes). Thus the WT KoRV-infected DERSE and 293T cells did not express glyco-gag.Fig. 3


Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms.

Nitta T, Ha D, Galvez F, Miyazawa T, Fan H - Retrovirology (2015)

Comparison of WT and putative glyco-gag-mutated KoRVs in viral production. a DERSE cells were infected with WT and glyco-gag-mutated (gg-) KoRVs produced from 293T cells transfected with pKoRV522 and pKoRV gg-. Gag in the cell lysates and media were detected by western blots using anti-KoRV CA antibodies. Western blotting for beta-Tubulin in the cell lysates confirmed equal loading of samples (not shown). b Cell lysates from 293T cells transfected with pKoRV522 or from the M-MuLV infected cell line 43D were treated with PNGase (endoglycosidase) F to remove N-linked oligosaccharides, and Gag proteins were detected by SDS-PAGE and western blots using anti-KoRV CA and anti-MuLV p30 antibodies. The locations of the Pr65gag Gag polyprotein precursor as well as a major cleavage product (Pr55gag) are shown, as well as the corresponding proteins (Pr60gag and Pr50gag) for KoRV. M-MuLV glyco-gag (the primary translation product gPr80gag as well as more slowly migrating forms with additional glycosylation) is indicated; endo F treatment reduced the size of gPr80gag to 75 kDa [48]. Virus release efficiencies of gg- KoRV compared to WT KoRV (set at 1 in each experiment) are shown for infected DERSE (c) and 293T (d) cells. The release efficiency measurements resulted from at least three independent experiments; error bars indicate standard deviation.
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Fig4: Comparison of WT and putative glyco-gag-mutated KoRVs in viral production. a DERSE cells were infected with WT and glyco-gag-mutated (gg-) KoRVs produced from 293T cells transfected with pKoRV522 and pKoRV gg-. Gag in the cell lysates and media were detected by western blots using anti-KoRV CA antibodies. Western blotting for beta-Tubulin in the cell lysates confirmed equal loading of samples (not shown). b Cell lysates from 293T cells transfected with pKoRV522 or from the M-MuLV infected cell line 43D were treated with PNGase (endoglycosidase) F to remove N-linked oligosaccharides, and Gag proteins were detected by SDS-PAGE and western blots using anti-KoRV CA and anti-MuLV p30 antibodies. The locations of the Pr65gag Gag polyprotein precursor as well as a major cleavage product (Pr55gag) are shown, as well as the corresponding proteins (Pr60gag and Pr50gag) for KoRV. M-MuLV glyco-gag (the primary translation product gPr80gag as well as more slowly migrating forms with additional glycosylation) is indicated; endo F treatment reduced the size of gPr80gag to 75 kDa [48]. Virus release efficiencies of gg- KoRV compared to WT KoRV (set at 1 in each experiment) are shown for infected DERSE (c) and 293T (d) cells. The release efficiency measurements resulted from at least three independent experiments; error bars indicate standard deviation.
Mentions: As mentioned in the introduction, the KoRV genome contains sequences that could potentially encode a typical glyco-gag. Examination of the KoRV (J group) RNA sequence indicated that KoRV has three potential upstream CUG codons in the same reading frame as the Gag polyprotein Pr60gag (Fig. 3). Moreover one protein sequence motif conserved in other gammaretroviral glyco-gags is present in the putative glyco-gag of KoRV: LGDVP at the N-terminus if initiation is at the CUG at nt 736. In addition a stretch of hydrophobic (potential membrane-spanning and/or signal peptide) amino acids is immediately upstream of the AUG for Pr60gag as for other gammaretroviral glyco-gags. There are three major KoRV isolates with different biological properties (KoRV-A, KoRV-B and KoRV-J) [18], and all of them showed nearly identical nucleic acid and protein sequences beginning with the conserved LGDVP motif in the leader peptide sequence (Additional file 1: Figs. S1, S2). To assess whether KoRV produces functional glyco-gag protein analogous to those in MuLVs, we introduced a mutation that would disrupt expression of putative glyco-gag protein in the plasmid containing the full-length KoRV molecular clone, pKoRV522 (Fig. 3); this plasmid was termed pKoRV gg-. WT and putative glyco-gag mutant KoRV stocks were prepared by transiently transfecting 293T cells with pKoRV522 and pKoRV gg-, and then used to infect DERSE or 293T cells. The infected cells were serially passaged until they all were infected, resulting in the stably infected cells DERSE/WT, DERSE/gg-, 293T/WT and 293T/gg-. As shown in Fig. 4a and quantified in Fig. 4c, the levels of Pr60gag in DERSE cells infected with WT and glyco-gag mutant KoRV were equivalent, as were the amounts of CA (virus) released into the media. Likewise 293T cells infected with the two viruses showed equivalent efficiencies of release (Fig. 4d). These results suggested that KoRV glyco-gag may not enhance virus release. On the other hand, western blots using anti-KoRV CA on the WT KoRV-infected cells did not show higher molecular weight proteins in addition to Pr60gag, which would possibly indicate that the infected DERSE and 293T cells did not express KoRV glyco-gag. In cells infected with WT MuLVs, glyco-gag proteins were sometimes somewhat diffuse in SDS-PAGE due to their glycosylation, and they migrated as more distinct and rapidly migrating bands if the glycosylation was removed [9]. As shown in Fig. 4b, treatment of cell extracts from WT M-MuLV infected cells with PNGase F (endoglycosidase F) showed a shift in mobility of the glyco-gag proteins (gPr80gagand gPr95gag-, right lanes). On the other hand, there was no evidence for equivalent proteins in the cells infected with WT KoRV (left lanes). Thus the WT KoRV-infected DERSE and 293T cells did not express glyco-gag.Fig. 3

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, 92697-3905, USA. nittat@savannahstate.edu.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus