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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

Restriction of KoRV replication by human and mouse APOBEC3s. 293T cells were cotransfected with pKoRV522 or pKoRV gg- along with plasmids expressing hA3G or mA3∆E5. The amount of CA released from the transfected 293T cells was measured by western blots. DERSE cells were infected with the resulting viruses with the similar inputs (measured by KoRV CA) and cell lysates of the infected DERSE cells were subjected to western blots with anti-KoRV CA (Additional file 1: Fig. S3). Relative infectivities of the different KoRV samples were assessed by quantifying the Gag signals by densitometry. The results were normalized for the amounts of the viruses used for infection. All values are shown relative to the infectivity of WT KoRV or KoRVgg- in the absence of any APOBEC3s. The experiments were repeated at least 3 times and the error bars indicate standard deviation (a, b). a The relative infectivity of KoRVgg- to KoRV522 is shown, where the infectivity of KoRV522 was set as 1. b The effects of co-transfecting different APOBEC3 expression plasmids on the infectivities of WT (pKoRV522) or KoRVgg- virus is shown. The APOBEC3 plasmids were co-transfected into the 293T cells at a 1:1 ratio with the KoRV expression plasmids. Relative viral infectivity to the control virus (without APOBECs) is shown. The experiments in (a) and (b) were repeated at least three times and the standard deviations are shown. The effects of co-transfecting different amounts of hA3G (c) or mA3∆E5 (d) with pKoRV522 in the 293T cells on the relative viral infectivity of WT KoRV in DERSE cells is shown. In d, it was not possible to obtain enough virus from the co-transfections at a 1:3 ratio for mA3∆E5 for the infectivity assay due to the inhibitory effect of this plasmid on KoRV expression (ND).
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Fig6: Restriction of KoRV replication by human and mouse APOBEC3s. 293T cells were cotransfected with pKoRV522 or pKoRV gg- along with plasmids expressing hA3G or mA3∆E5. The amount of CA released from the transfected 293T cells was measured by western blots. DERSE cells were infected with the resulting viruses with the similar inputs (measured by KoRV CA) and cell lysates of the infected DERSE cells were subjected to western blots with anti-KoRV CA (Additional file 1: Fig. S3). Relative infectivities of the different KoRV samples were assessed by quantifying the Gag signals by densitometry. The results were normalized for the amounts of the viruses used for infection. All values are shown relative to the infectivity of WT KoRV or KoRVgg- in the absence of any APOBEC3s. The experiments were repeated at least 3 times and the error bars indicate standard deviation (a, b). a The relative infectivity of KoRVgg- to KoRV522 is shown, where the infectivity of KoRV522 was set as 1. b The effects of co-transfecting different APOBEC3 expression plasmids on the infectivities of WT (pKoRV522) or KoRVgg- virus is shown. The APOBEC3 plasmids were co-transfected into the 293T cells at a 1:1 ratio with the KoRV expression plasmids. Relative viral infectivity to the control virus (without APOBECs) is shown. The experiments in (a) and (b) were repeated at least three times and the standard deviations are shown. The effects of co-transfecting different amounts of hA3G (c) or mA3∆E5 (d) with pKoRV522 in the 293T cells on the relative viral infectivity of WT KoRV in DERSE cells is shown. In d, it was not possible to obtain enough virus from the co-transfections at a 1:3 ratio for mA3∆E5 for the infectivity assay due to the inhibitory effect of this plasmid on KoRV expression (ND).

Mentions: To assess the effects of the different APOBEC3s on KoRV, infectivity assays were performed in DERSE cells. The cells were infected with viruses collected from 293T cells transiently transfected with pKoRV522 or pKoRV gg- (to test for the effects of glyco-gag) along with the plasmids expressing APOBEC3s. DERSE cells were infected with media from the different transfections, and levels of infection were determined by SDS-PAGE and western blotting of cell extracts with anti-KoRV CA antiserum. As shown in Fig. 6a, infectivities of WT and gg- KoRVs were comparable. Expression of hA3G and mA3∆E5 strongly impaired KoRV infection (95–98% reduction, Fig. 6b; Additional file 1: Fig. S3). The abilities of hA3G and mA3∆E5 to restrict KoRV infection were dose-dependent and equivalent in strength (Fig. 6c, d). The equivalent restriction of WT and gg- KoRVs by the APOBEC3s was consistent with the lack of detectable expression of glyco-gag in WT KoRV-infected cells (Fig. 4).Fig. 6


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

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

Restriction of KoRV replication by human and mouse APOBEC3s. 293T cells were cotransfected with pKoRV522 or pKoRV gg- along with plasmids expressing hA3G or mA3∆E5. The amount of CA released from the transfected 293T cells was measured by western blots. DERSE cells were infected with the resulting viruses with the similar inputs (measured by KoRV CA) and cell lysates of the infected DERSE cells were subjected to western blots with anti-KoRV CA (Additional file 1: Fig. S3). Relative infectivities of the different KoRV samples were assessed by quantifying the Gag signals by densitometry. The results were normalized for the amounts of the viruses used for infection. All values are shown relative to the infectivity of WT KoRV or KoRVgg- in the absence of any APOBEC3s. The experiments were repeated at least 3 times and the error bars indicate standard deviation (a, b). a The relative infectivity of KoRVgg- to KoRV522 is shown, where the infectivity of KoRV522 was set as 1. b The effects of co-transfecting different APOBEC3 expression plasmids on the infectivities of WT (pKoRV522) or KoRVgg- virus is shown. The APOBEC3 plasmids were co-transfected into the 293T cells at a 1:1 ratio with the KoRV expression plasmids. Relative viral infectivity to the control virus (without APOBECs) is shown. The experiments in (a) and (b) were repeated at least three times and the standard deviations are shown. The effects of co-transfecting different amounts of hA3G (c) or mA3∆E5 (d) with pKoRV522 in the 293T cells on the relative viral infectivity of WT KoRV in DERSE cells is shown. In d, it was not possible to obtain enough virus from the co-transfections at a 1:3 ratio for mA3∆E5 for the infectivity assay due to the inhibitory effect of this plasmid on KoRV expression (ND).
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Fig6: Restriction of KoRV replication by human and mouse APOBEC3s. 293T cells were cotransfected with pKoRV522 or pKoRV gg- along with plasmids expressing hA3G or mA3∆E5. The amount of CA released from the transfected 293T cells was measured by western blots. DERSE cells were infected with the resulting viruses with the similar inputs (measured by KoRV CA) and cell lysates of the infected DERSE cells were subjected to western blots with anti-KoRV CA (Additional file 1: Fig. S3). Relative infectivities of the different KoRV samples were assessed by quantifying the Gag signals by densitometry. The results were normalized for the amounts of the viruses used for infection. All values are shown relative to the infectivity of WT KoRV or KoRVgg- in the absence of any APOBEC3s. The experiments were repeated at least 3 times and the error bars indicate standard deviation (a, b). a The relative infectivity of KoRVgg- to KoRV522 is shown, where the infectivity of KoRV522 was set as 1. b The effects of co-transfecting different APOBEC3 expression plasmids on the infectivities of WT (pKoRV522) or KoRVgg- virus is shown. The APOBEC3 plasmids were co-transfected into the 293T cells at a 1:1 ratio with the KoRV expression plasmids. Relative viral infectivity to the control virus (without APOBECs) is shown. The experiments in (a) and (b) were repeated at least three times and the standard deviations are shown. The effects of co-transfecting different amounts of hA3G (c) or mA3∆E5 (d) with pKoRV522 in the 293T cells on the relative viral infectivity of WT KoRV in DERSE cells is shown. In d, it was not possible to obtain enough virus from the co-transfections at a 1:3 ratio for mA3∆E5 for the infectivity assay due to the inhibitory effect of this plasmid on KoRV expression (ND).
Mentions: To assess the effects of the different APOBEC3s on KoRV, infectivity assays were performed in DERSE cells. The cells were infected with viruses collected from 293T cells transiently transfected with pKoRV522 or pKoRV gg- (to test for the effects of glyco-gag) along with the plasmids expressing APOBEC3s. DERSE cells were infected with media from the different transfections, and levels of infection were determined by SDS-PAGE and western blotting of cell extracts with anti-KoRV CA antiserum. As shown in Fig. 6a, infectivities of WT and gg- KoRVs were comparable. Expression of hA3G and mA3∆E5 strongly impaired KoRV infection (95–98% reduction, Fig. 6b; Additional file 1: Fig. S3). The abilities of hA3G and mA3∆E5 to restrict KoRV infection were dose-dependent and equivalent in strength (Fig. 6c, d). The equivalent restriction of WT and gg- KoRVs by the APOBEC3s was consistent with the lack of detectable expression of glyco-gag in WT KoRV-infected cells (Fig. 4).Fig. 6

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