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. 2019 Dec 12;15(12):e1008173.
doi: 10.1371/journal.ppat.1008173. eCollection 2019 Dec.

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing

Yoshiyuki Hakata  1 , Jun Li  1   2 , Takahiro Fujino  3 , Yuki Tanaka  3 , Rie Shimizu  1 , Masaaki Miyazawa  1   4
Affiliations

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing

Yoshiyuki Hakata et al. PLoS Pathog. .

Abstract

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Infectious F-MuLV molecular clones and their protease-deficient mutants.
(A) A diagrammatic alignment of p57(2LTR)Sp72 (57) and GB57 sequences. The latter is enrolled in GenBank (accession number X02794). Discrepancies are indicated by arrowheads and corresponding nucleotide position numbers. Sequences in between these discrepant residues are omitted and represented by filled circles. Mutants (1M to 6M and 57pr) were generated with the strain 57 sequence as the backbone with the indicated GB57-type nucleotide substitutions. The strain 57 sequence in p57(2LTR)Sp72 has been registered in DDBJ with the accession number LC229035. (B) Infectivity of strain 57 and 6M viruses. Viruses were produced from 293T cells transfected with either one of the virus-encoding plasmids. As a control (ctrl), the cells were mock-transfected with a control vacant plasmid. A portion of the culture supernatant was used for the infectivity assay in which virus infectivity was evaluated by counting the number of gp70-positive foci on Mus dunni cells. Results are shown as a mean with standard error of more than 3 wells. *, P = 0.0004 by Student’s t-test. Experiments were repeated three times with similar results. The remaining virus-containing supernatant was subject to ultracentrifugation to collect the viruses. The pelleted viruses and the transfected 293T cells were lysed for immunoblotting. To detect the matrix protein (p15) and its precursor, Pr65gag, the anti-p15 mAb (clone 690) was used. The envelope protein (gp70) was also detected to monitor virus production using the anti-F-MuLV gp70 mAb (clone 720). Actin is used as a loading control for the cells. (C) Detection of p15 in mutant virus particles. Viruses were produced by transient transfection and collected from the culture supernatant, and analyzed as described for panel B. The cell lysates were also analyzed as described for panel B. (D) Variations in amino acid residue at position 96 (indicated by the arrowhead) within MuLV protease. For HIV, the amino acid residue indicated by the arrowhead is located at position 88 of viral protease. FB29, GenBank accession number Z11128; XMRV, GenBank accession number NC_007815.1; HIV-1 strain NL4-3, GenBank accession number M19921. (E) A single nucleotide substitution at position 2509 that results in the D-to-H amino acid substitution at position 96 of the viral protease inactivates Pr65gag processing in F-MuLV strain 57. The mutant, 57pr, harbors the D-to-H substitution at position 96 of the protease. Viruses were produced by transient transfection and collected by ultracentrifugation. The virus and cell lysates were subjected to immunoblotting. Anti-p15 (MA) mAb clone 690, anti-F-MuLV gp70 (SU) mAb clone 720 and anti-actin Ab C-11 were used to detect p15 and Pr65gag, gp70, and cellular actin, respectively. (F) The same analyses as shown in panel E except that FB29 and its mutant (FB29pr) which harbors the D-to-H substitution at position 96 of the protease were used. A faint band discernible close to the molecular weight of Pr65gag in the control lane is a non-specific signal.
Fig 2
Fig 2. mA3 interacts with F-MuLV protease.
(A) 293T cells were transfected with 6 μg of F-MuLV 57- or 57pr-encoding plasmid along with 1.5 μg of the FLAG-tagged mA3 (5+ or Δ5) expression one or the control (−) parental pCMV2-FLAG plasmid. Viruses and the cells were collected at the indicated time-points after transfection, and analyzed by immunoblotting. Anti-p15 (MA) mAb clone 690, anti-F-MuLV gp70 (SU) mAb clone 720, and anti-actin Ab C-11 were used to detect Pr65gag, gp70, and cellular actin, respectively. To detect mA3, the anti-FLAG mAb M2 was used. (B) The same experiments were performed as described for panel A except that FB29 and FB29pr were used. (C) The enzymatically active WT viral protease (PR) or the inactive G2509C mutant protein (PR (G2509C)) from strain 57 were prepared by using the in vitro transcription/translation system (input). Both proteins were initially expressed with twenty amino acid residues flanking each end of the protease. Since the flanking peptides of 20PR20 were autocatalytically cleaved by active PR, the processed mature viral protease migrated to produce a signal at around 15kDa. On the other hand, the mutant 20G/C20 is unable to cut the flanking peptides, resulting in the band with a molecular mass higher than that of mature viral protease. For the control (−), no DNA was added in the reaction. For the pull-down assays, GST or GST-conjugated Δ5 mA3 bound on Glutathione Sepharose 4B was mixed with the above expressed PR or 20G/C20. The associated protease in the pulled-down fraction was detected by immunoblotting using the rabbit anti-MuLV protease Ab we prepared. For the detection of GST-conjugated proteins, the anti-GST Ab was used. (D) Deletion mutants of strain 57 protease were synthesized by the in vitro transcription/translation system. These were mixed with GST, GST-conjugated Δ5, or GST-conjugated 5+ mA3 bound on Glutathione Sepharose 4B. The associated protease or its fragments were detected by immunoblotting as described for panel C.
Fig 3
Fig 3. Virus-incorporated mA3 perturbs autocatalytic Pr180gag-pol cleavage and inhibits Pr65gag processing.
(A) A comparison of Pr65gag processing patterns in the presence and absence of mA3. Strain 57 viruses were generated in the presence (5+ or Δ5) or absence (−) of mA3. The virions and cell lysates were analyzed by immunoblotting. Anti-p15 (MA) mAb clone 690, anti-F-MuLV gp70 (SU) mAb clone 720, anti-FLAG mAb M2, and anti-actin Ab C-11 were used to visualize p15 and Pr65gag, gp70, mA3, and cellular actin, respectively. Band intensities of p15 and Pr65gag on the same blot were measured and p15/Pr65gag ratios were calculated. The data represent means with standard errors from three independent experiments. *, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests. (B) The same experiments as described for panel A except that FB29 was used. *, P < 0.001; #, P < 0.05 by one-way ANOVA with Tukey’s multiple comparison tests. (C and D) The same virus lysates were used as shown in panels A and B to detect mature viral protease and its precursor, Pr180gag-pol. The rabbit anti-MuLV protease Ab was used for immunoblotting. The image taken with a long exposure time for the detection of mature viral protease within FB29 virions was also shown. Band intensities of mature viral protease and Pr180gag-pol were measured on the same blot, and PR/Pr180gag-pol ratios were calculated. The data represent means with standard errors from three independent experiments. *, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests.
Fig 4
Fig 4. Physiologically relevant amounts of mA3 suffice for the inhibition of Pr65gag processing and perturbation of Pr180gag-pol autoprocessing.
(A) MEF cells of WT (W) and mA3 KO (K) B6 mice were infected with F-MuLV FB29 (+) or mock infected (−). At a day after infection, the cells were treated with LPS. At 3 days postinfection, produced viruses and the cells were harvested and analyzed by immunoblotting. Anti-p15 (MA) mAb clone 690, anti-F-MuLV gp70 (SU) mAb clone 720, and anti-actin Ab C-11 were used to detect p15 and Pr65gag, gp70, and cellular actin, respectively. For the detection of PR and Pr180gag-pol, the rabbit anti-MuLV protease Ab was used. The anti-mA3 Ab was pre-absorbed with mA3 KO spleen extract, and used for the detection of endogenous mA3. Pr65gag signals detectable after a long exposure time are also shown. (B) Band intensities of p15 and Pr65gag on the same blot as shown in the middle part of panel A were measured, and p15/Pr65gag ratios were calculated and expressed by setting the ratio for the KO sample as 100 for each experiment. The data represent the mean with standard error from three independent experiments. *, P = 0.0015 by one-sample t-test comparing to the value 100. (C) Band intensities of mature viral protease and Pr180gag-pol on the same blot as shown in the right part of panel A were measured and PR/Pr180gag-pol ratios were calculated as above. The data represent the mean with standard error from three independent experiments. *, P = 0.0035 by one-sample t-test comparing to the value 100.
Fig 5
Fig 5. The impact of mA3 on viral protease-mediated processes was deaminase-independent.
(A-C) The experiments were performed similarly to those shown in Fig 3A and 3C except by using the deaminase-inactivated E73A mutant (Δ5 E73A) along with the wild-type Δ5 mA3. The data represent means with standard errors from three independent experiments. *, P < 0.001; #, P < 0.01 by one-way ANOVA with Tukey’s multiple comparison tests. (D-F) The experiments were performed similarly to those shown in Fig 3B and 3D except by using the E73A mutant along with the wild-type Δ5 mA3. The image taken after a long exposure time for the detection of mature viral protease is also shown. *, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests.
Fig 6
Fig 6. The effect of C-terminal half of mA3 on Pr65gag processing and autocatalytic Pr180gag-pol cleavage.
(A-C) The experiments were performed similarly to those shown in Fig 3A and 3C except by including the C-terminal half of Δ5 mA3 (Δ5Ch). The data represent means with standard errors from three independent experiments. *, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison tests.
Fig 7
Fig 7. mA3 binds to Pr180gag-pol mainly through its C-terminal half and reduces mature viral protease.
(A) Assays for Pr180gag-pol autoprocessing using the in vitro transcription and translation (IVT) system. No template DNA (−) or 1 μg of pT7CFE1-Pr180gag-pol(57) (+) was added to the IVT reaction. The reaction mixture was incubated for the indicated duration at 30°C, reaction stopped by the addition of Laemmli SDS sample buffer, and the expressed proteins analyzed by immunoblotting using the rabbit anti-MuLV protease Ab. (B) The effect of Δ5 mA3 on Pr180gag-pol autoprocessing analyzed by using the IVT system. Into the IVT reaction 0.38 μg of pT7CFE1-Pr180gag-pol(57) was added along with 0.26, 0.17 or 0.09 μg of pT7CFE1-Δ5mA3. The mixture was incubated for 20 hours at 30°C. The lysates were analyzed by immunoblotting. To detect Δ5 mA3, the anti-FLAG mAb was used as it is expressed as tagged protein. The rabbit anti-MuLV protease Ab was used to detect PR and Pr180gag-pol. The band intensities of PR and Pr180gag-pol on the same blot were measured and PR/Pr180gag-pol ratios were calculated (the bar chart). The data represent means with standard errors from three independent experiments. *, P < 0.001 for the three indicated comparisons; #, P < 0.01 by one-way ANOVA with Tukey’s multiple comparison tests. (C) Into the IVT reaction, 0.38 μg of pT7CFE1-Pr180gag-pol(57) was added (+) along with 0.2μg of pCFE-GFP control (GFP), pT7CFE1-Δ5 mA3 (Δ5), pT7CFE1-Δ5 mA3 N-half (Δ5Nh) or pT7CFE1-Δ5 mA3 C-half (Δ5Ch) expression plasmid. The mixture was incubated for 20 hours at 30°C. A portion of the reaction mixture was analyzed by immunoblotting (IVT). The image taken after a long exposure time is also shown in the bottom. Another portion of the mixture was used for the immunoprecipitation assay using the anti-FLAG Ab to precipitate FLAG-tagged mA3, and the precipitates were analyzed by immunoblotting to detect bound protease or its precursor (IP). The rabbit anti-MuLV protease Ab was used to detect PR and Pr180gag-pol. Anti-FLAG mAb M2 were used to detect flag-tagged mA3 proteins.
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This work was supported by Japan Society for the Promotion of Sciences (JSPS: https://www.jsps.go.jp) Grants-in-Aid for Scientific Research (KAKENHI) numbers 15H01268 (MM), 16H05199 (MM), and 16K08821 (YH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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