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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

Titration of KoRV infection with DERSE cells. DERSE cells were infected with different doses of KoRV (μl of 293T cell supernatant) as in Fig. 1. Fluorescence for GFP-positive cells is shown in a) and SDS-PAGE and western blotting for KoRV Gag and GFP is shown in b. The Gag polyprotein precursor Pr60gag is indicated, as well as a major proteolytic cleavage product Pr50gag.
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Fig2: Titration of KoRV infection with DERSE cells. DERSE cells were infected with different doses of KoRV (μl of 293T cell supernatant) as in Fig. 1. Fluorescence for GFP-positive cells is shown in a) and SDS-PAGE and western blotting for KoRV Gag and GFP is shown in b. The Gag polyprotein precursor Pr60gag is indicated, as well as a major proteolytic cleavage product Pr50gag.

Mentions: Detection of KoRV infection in DERSE cells. DERSE cells were infected with KoRV released from 293T cells transiently transfected with pKoRV522, and they showed GFP expression as indicated by fluorescence microscopy (middle of the figure), indicative of KoRV replication. Supernatants from the infected DERSE cells (after 5 passages) were used to secondarily infect fresh 293T cells (bottom of the figure). The infected 293T cells were green and SDS-PAGE and western blot for KoRV Gag protein (bottom right) showed that the cultures were expressing KoRV Gag protein, indicative of productive infection. Uninfected 293T and DERSE cells (see Fig. 2) showed no background green fluorescence.


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

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

Titration of KoRV infection with DERSE cells. DERSE cells were infected with different doses of KoRV (μl of 293T cell supernatant) as in Fig. 1. Fluorescence for GFP-positive cells is shown in a) and SDS-PAGE and western blotting for KoRV Gag and GFP is shown in b. The Gag polyprotein precursor Pr60gag is indicated, as well as a major proteolytic cleavage product Pr50gag.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528783&req=5

Fig2: Titration of KoRV infection with DERSE cells. DERSE cells were infected with different doses of KoRV (μl of 293T cell supernatant) as in Fig. 1. Fluorescence for GFP-positive cells is shown in a) and SDS-PAGE and western blotting for KoRV Gag and GFP is shown in b. The Gag polyprotein precursor Pr60gag is indicated, as well as a major proteolytic cleavage product Pr50gag.
Mentions: Detection of KoRV infection in DERSE cells. DERSE cells were infected with KoRV released from 293T cells transiently transfected with pKoRV522, and they showed GFP expression as indicated by fluorescence microscopy (middle of the figure), indicative of KoRV replication. Supernatants from the infected DERSE cells (after 5 passages) were used to secondarily infect fresh 293T cells (bottom of the figure). The infected 293T cells were green and SDS-PAGE and western blot for KoRV Gag protein (bottom right) showed that the cultures were expressing KoRV Gag protein, indicative of productive infection. Uninfected 293T and DERSE cells (see Fig. 2) showed no background green fluorescence.

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