Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.


Introduction
During long co-evolutionary history, viral pathogens keep developing increasingly novel weapons which against hosts to facilitate viral survival and pathogenesis, via encoding not only structural and enzymatic proteins but also various accessory proteins where required. Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and other primate lentiviruses encode accessory proteins that enhance viral infectivity [1]. These include Viral protein R (Vpr), Viral infectivity factor (Vif), Viral protein U (Vpu), and negative regulation factor (Nef). In addition, some subsets of primate

Cell Culture, Transfection, and Drug Treatment
Human embryonic kidney HEK293T cells, HEK293, and Human cervical HeLa cells were maintained in Dulbecco's Modified Eagle Medium (Gibco, Beijing, China) supplemented with 10% fetal calf serum in a 5% CO 2 incubator at 37 • C. Plasmid transfection was performed using FuGENE HD (Promega, Madison, WI, USA). For the experiment involving proteasome reversible inhibitor MG132 (Sigma-Aldrich, St. Louis, MO, USA) and irreversible inhibitor Lactacystin (EMD Millipore, Darmstadt, Germany), cells were transfected with the indicated plasmids for 43 h before addition of the inhibitor and cultured for a further 5 h.

Immunofluorescence Staining
HeLa cells (2.5 × 10 5 ) or HEK293 cells (2.5 × 10 5 ) were seeded on cover glasses in a 12-well plate and transfected with 1 µg of pcDNA 3.1/HA-MCM10, with or without 1 µg of pcDNA3.1/3 × FLAG-Vpr. Following 48 h of transfection, immunofluorescence staining was performed as described previously [24]. In brief, cells on a cover slip were fixed with 4% paraformaldehyde for 10 min at room temperature. Paraformaldehyde was then replaced with cold methanol and the cells were maintained at −20 • C for 20 min. The cells were then washed with PBS and incubated with anti-FLAG rabbit mAb (MBL), anti-HA mouse mAb (MBL), or anti-gamma H2AX mouse mAb (Abcam, Cambridge, UK) for 1 h at room temperature. Following further washing with PBS, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) or Alexa Fluor 594 goat anti-mouse IgG (Invitrogen) was added for 1 h at room temperature in the dark. Nucleus was stained with Hoechst 33342 (Thermo Fisher Scientific) for 5 min in the dark. Coverslips were then rinsed with PBS and mounted on glass slides. Processed samples were visualized using a confocal fluorescence microscope (IX81-FV1000-D/FLUOVIEW System, Olympus, Tokyo, Japan).

Cell Cycle Analysis
HeLa cells (1 × 10 5 ) were seeded in a 6-well plate and transfected with 2 µg of pME18neo/FLAG-Vpr-IRESZsGreen1 Vpr wild type or the panel of mutants stated above. After 48 h, the cells were harvested and fixed using 70% ethanol. After being washed twice with PBS, the cells were resuspended in RNase A (Invitrogen; 100 µg/mL) at 37 • C for 20 min and stained with propidium iodide (PI; Sigma; 50 µg/mL) at room temperature for 10 min. Stained cells were analyzed using a BD Accuri TM C6 Plus with a sampler flow cytometer (Becton-Dickinson, Franklin lakes, NJ, USA). The data were analyzed using FlowJo v10 (FlowJo, LLC, Ashland, OR, USA).

Real-Time qRT-PCR Analysis of Human MCM10 mRNA Expression
Total RNA was extracted using the RNeasy mini kit with DNase digestion, according to the manufacturer's instructions (QIAGEN). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher) and stored at −80 • C until use. Reverse transcription was performed using High Capacity RNA-to-cDNA (Thermo Fisher), according to the manufacturer's manual. qRT-PCR was performed using a Prism 7500FAST sequence detection system (Applied Biosystems). Samples were run in triplicate and all data were normalized to GAPDH mRNA expression as an internal control.

Statistical Analysis
All data were expressed as mean ± standard deviation, based on at least 3 independent experiments. Statistical significance was evaluated using Student's t-test. Differences were estimated to be significant at p < 0.05 (*), and strongly significant at p < 0.01 (**) and p < 0.001(***).
Correlation efficient was analyzed with linear regression and significance was calculated with Pearson correlation analysis.
Overall MCM10 down-regulation profiles and primate lentiviruses classification were comprehensively analyzed. Interestingly, all Vpr proteins belonging to prototype viruses did not enhance the downregulation of MCM10, while some HIV-1 (HIV-1 Vpr) and HIV-2 (SIVmus and SIVrcm Vpr) type viruses curbed MCM10 expression by varying degrees. However, no functional residues affecting MCM10 down-regulation were found, implying that there may be more than one residue from different sites corporately contributed to decreasing MCM10.   Structural analysis revealed that full length Vpr forms three amphipathic alpha helices surrounding a hydrophobic core (α-helix 1, 2, and 3) [25][26][27]. It also has a flexible, negatively charged N terminal domain flanking the helices, while its C-terminal domain is also flexible, positively charged, and rich in arginine residues. Based on the phylogeny of primate lentiviruses and virus type classification, amino acid sequences of 10 Vpr proteins from each group were analyzed using multiple alignments via the MEGA 7 program (HIV-2 Rod10 Vpx was added as the external reference). Then structure alignment was re-generated by ESPript 3.0 with HIV-1 Vpr structure as the criterion ( Figure 1B). Sequence alignments indicated that all Vpr/Vpx proteins shared conserved tertiary structures. For example, residues framed by a blue-line box depicted similarities in both sequence and structure. Additionally, such similarities were mainly enriched in three α-helices, including the residues 18-34, 38-49, and 54-77. Interestingly, all lentiviral Vpr proteins displayed potential zinc-binding motifs (H33, H71, H76, and *78) located in α-helix 2 and 3, which were similar to the conserved HIV-2 Vpx zinc-binding motif (HHCC). It was suggested that a zinc-binding motif is essential for maintaining both Vpr and Vpx. The potential zinc-binding motif of Vpr/Vpx also engaged E3 ubiquitin ligase complex formation and hijacked it to induce cellular factor degradation [28]. By contrast, flexible C-terminal domains exhibited sequence diversity compared to the central region of Vpr/Vpx proteins. Typically, HIV-2 Vpx was characterized by a poly-proline motif (PPM) in the C-terminal domain (residues from 91 to 97) whereas other lentiviruses Vpr proteins possessed few such properties.
Next, in order to compare the expression and function of these Vpr/x proteins, 10 HIV/SIV Vpr and 1 Vpx were synthesized according to nucleotide sequences collected as stated above. Subsequently, HEK293T cells were transfected with pcDNA3.1 that encoded 3 × FLAG-tagged HIV/SIV Vpr/x proteins, namely, 3 × FLAG-HIV-1, SIVdeb, SIVsyk, SIVlst, SIVmus, SIVmon, SIVrcm, SIVagm, SIVmac, and SIVcol Vprs, and HIV-2 Vpx, or the control, pcDNA3.1/3 × FLAG and examined expression via western blotting with the anti-FLAG-mAb. Specific expression levels of all the 11 HIV/SIV Vpr and Vpx were detectable via western blotting ( Figure 1C). We next verified sub-cellular distribution of all the 11 primate Vprs by immunofluorescence staining in Hela cells. For all Vpr proteins, dominant distribution of Vpr in nucleus of HeLa cells was observed [29]. Such results are coherent with previous works.

MCM10 down-Regulation by Primate Lentiviruses Vpr/x Proteins
In order to verify the changes in MCM10 expression caused by various HIV/SIV Vpr/Vpx proteins, co-transfection with pcDNA 3.1/HA-MCM10 and pcDNA3.1/3 × FLAG-HIV/SIV Vpr/x was carried out in HEK293T cells and HA-MCM10 expression was monitored via western blotting (Figure 2A). Densities of the HA-MCM10 band were normalized with those of tubulin. The relative density of HA-MCM10 was decreased by co-transfection of HIV-1, SIVmus, and SIVrcm Vpr proteins (Figure 2A lower panel), while other strains failed to induce similar down-regulation of MCM10. MCM10 expression due to the presence of HIV-1 Vpr protein decreased to approximately 62% compared to only-MCM10 control. In addition, MCM10 expression decreased to 40% and 54% due to the presence of SIVmus and SIVrcm Vpr, respectively. In contrast, HIV-2 Vpx did not downregulate MCM10.
Furthermore, we investigated whether endogenous MCM10 protein expression levels were also susceptible to HIV-1, SIVmus, and SIVrcm Vpr proteins. Likewise, endogenous MCM10 degradation were validated ( Figure 2C). MCM10 expression was downregulated to 62%, 79%, and 63% compared to only-MCM10 control, respectively ( Figure 2C right panel). Next, we performed real-time qRT-PCR analysis of MCM10 mRNA expression in HEK293T cells, which were transiently transfected with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus, or SIVrcm Vprs and found that MCM10 mRNA expression was detected in similar levels among transfections of HIV-1, SIVmus, and SIVrcm Vpr proteins. This result indicated that down-regulation of endogenous MCM10 by HIV-1, SIVmus, and SIVrcm were induced at protein level ( Figure 2D).  and α-Tubulin were quantified by densitometry analysis using ImageJ software. The relative intensities were calculated as the ratio of density of MCM10 to density of α-Tubulin. Each column and error bar represents mean ± SD for three independent experiments (right panel). The asterisk indicates a statistically significant difference (* p < 0.05, ** p < 0.01). (D) Samples were run in triplicate and all data were normalized to GAPDH mRNA expression as an internal control.

MCM10 Degradation via Proteasome Dependent Pathway
In order to verify whether MCM10 degradation by distinct HIV-1, SIVmus, and SIVrcm Vpr proteins resulted via the proteasomal degradation pathway, MG-132, a reversible proteasome inhibitor, was used to monitor the effects on MCM10 expression. At 43 h following co-transfection of pcDNA3.1/HA-MCM10 together with pcDNA3.1/3 × FLAG-HIV-1, SIVmus, and SIVrcm Vprs or the control pcDNA3.1/3 × FLAG, HEK293T cells were treated with 10 μM MG-132 or DMSO. At 48 h after Band densities of endogenous MCM10 and α-Tubulin were quantified by densitometry analysis using ImageJ software. The relative intensities were calculated as the ratio of density of MCM10 to density of α-Tubulin. Each column and error bar represents mean ± SD for three independent experiments (right panel). The asterisk indicates a statistically significant difference (* p < 0.05, ** p < 0.01). (D) Samples were run in triplicate and all data were normalized to GAPDH mRNA expression as an internal control.
Overall MCM10 down-regulation profiles and primate lentiviruses classification were comprehensively analyzed. Interestingly, all Vpr proteins belonging to prototype viruses did not enhance the downregulation of MCM10, while some HIV-1 (HIV-1 Vpr) and HIV-2 (SIVmus and SIVrcm Vpr) type viruses curbed MCM10 expression by varying degrees. However, no functional residues affecting MCM10 down-regulation were found, implying that there may be more than one residue from different sites corporately contributed to decreasing MCM10.

MCM10 Degradation via Proteasome Dependent Pathway
In order to verify whether MCM10 degradation by distinct HIV-1, SIVmus, and SIVrcm Vpr proteins resulted via the proteasomal degradation pathway, MG-132, a reversible proteasome inhibitor, was used to monitor the effects on MCM10 expression. At 43 h following co-transfection of pcDNA3.1/HA-MCM10 together with pcDNA3.1/3 × FLAG-HIV-1, SIVmus, and SIVrcm Vprs or the control pcDNA3.1/3 × FLAG, HEK293T cells were treated with 10 µM MG-132 or DMSO. At 48 h after transfection, HEK293T cells were harvested for western blotting. HIV-1, SIVmus, and SIVrcm Vpr proteins recovered MCM10 expression in MG-132-treated cultures, as compared with that of DMSO-treated cultures ( Figure 3A). By contrast, MG-132 exerted no effect on MCM10 expression in cells transfected with only the control, pcDNA3.1/3 × FLAG. This result was confirmed with another irreversible proteasome inhibitor, lactacystin, which also targets the 20S proteasome resulting in proteasomal degradation inhibition. Treatment with lactacystin (20 µM) resulted in an increase in MCM10 expression that was higher than the increase caused by MG 132 (Figure 3B), indicating that lactacystin is an irreversible inhibitor and its activity is more specific as compared with reversible inhibitor MG132, and thus the effect of MG132 may be smaller than that of lactacystin.
Viruses 2020, 12, x FOR PEER REVIEW 9 of 20 transfection, HEK293T cells were harvested for western blotting. HIV-1, SIVmus, and SIVrcm Vpr proteins recovered MCM10 expression in MG-132-treated cultures, as compared with that of DMSOtreated cultures ( Figure 3A). By contrast, MG-132 exerted no effect on MCM10 expression in cells transfected with only the control, pcDNA3.1/3 × FLAG. This result was confirmed with another irreversible proteasome inhibitor, lactacystin, which also targets the 20S proteasome resulting in proteasomal degradation inhibition. Treatment with lactacystin (20 μM) resulted in an increase in MCM10 expression that was higher than the increase caused by MG 132 (Figure 3B), indicating that lactacystin is an irreversible inhibitor and its activity is more specific as compared with reversible inhibitor MG132, and thus the effect of MG132 may be smaller than that of lactacystin.
To confirm this interaction, we performed an immunofluorescence assay in HeLa cells. HeLa cells were transiently transfected with pcDNA 3.1/HA-MCM10 together with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus and SIVrcm Vprs, or the control pcDNA3.1/3 × FLAG, and cells were stained with anti-FLAG mAb followed by Alexa Fluor 488 goat anti-rabbit IgG to detect Vpr (green), with anti-HA mAb followed by Alexa Fluor 594 goat anti-mouse IgG to detect MCM10 (red) and with Hoechst 33,342 to detect nucleus (blue) ( Figure 4B). HA-MCM10 was predominantly concentrated in the nucleus as a form of discrete replication foci. HIV-1, SIVmus, and SIVrcm Vpr expression also accumulated mostly in nucleus of HeLa cells. On the other hand, Vpr distribution was observed to aggregate in a dispersed form around the nucleoli. Vpr proteins partially colocalized with the MCM10 foci in the nucleus in merged images (orange) ( Figure 4B). Band densities of HA-MCM10 and α-Tubulin were quantified by densitometry analysis using ImageJ software. The relative intensities were calculated as the ratio of density of MCM10 to density of α-Tubulin.
To confirm this interaction, we performed an immunofluorescence assay in HeLa cells. HeLa cells were transiently transfected with pcDNA 3.1/HA-MCM10 together with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus and SIVrcm Vprs, or the control pcDNA3.1/3 × FLAG, and cells were stained with anti-FLAG mAb followed by Alexa Fluor 488 goat anti-rabbit IgG to detect Vpr (green), with anti-HA mAb followed by Alexa Fluor 594 goat anti-mouse IgG to detect MCM10 (red) and with Hoechst 33,342 to detect nucleus (blue) ( Figure 4B). HA-MCM10 was predominantly concentrated in the nucleus as a form of discrete replication foci. HIV-1, SIVmus, and SIVrcm Vpr expression also accumulated mostly in nucleus of HeLa cells. On the other hand, Vpr distribution was observed to aggregate in a dispersed form around the nucleoli. Vpr proteins partially colocalized with the MCM10 foci in the nucleus in merged images (orange) ( Figure 4B).

MCM 2-7 Interaction Region of MCM10 Susceptible to Degradation by Vprs
MCM10 is composed of different domains as follows: N-terminal domain (NTD, amino acids 1-145) responsible for MCM10 self-oligomerization, internal domains (ID, amino acids 230-427) interacting with proliferating cell nuclear antigen (PCNA) and DNA polymerase-α (Pol-α), C-terminal domain (CTD, amino acids 596-860) that interacts with DNA and polymerase-α, and MCM 2-7 interaction region (amino acids 530-655) mediating MCM10 interaction with MCM 2-7 complex, which is also essential for MCM10 nuclear localization ( Figure 5A) [21]. To investigate the determinant domain of MCM10 susceptible to degradation by Vpr proteins, pcDNA3.1 expression vectors encoding HA tagged domain-deficient mutants of MCM10, namely, HA-1-165, 1-427, 1-530, and 1-655 were constructed ( Figure 5A) and transfected into HEK293T cells. Then, the expression and distribution of these were examined by western blotting and immunofluorescence staining, respectively ( Figure 5B,C). The expression of all domain-deficient mutants of MCM10 was specifically detectable via western blotting using anti-HA mAb ( Figure 5B). Wildtype MCM10 aggregated in the nucleus and formed typical replication foci ( Figure 5C). By contrast, MCM10 (1-655) distribution detected in the cytoplasm was possibly caused by a lack of unidentified nuclear localization signals (NLSs) in CTD. Besides, a small fraction of MCM10 (1-655) was still localized in the nucleus but typical foci formation disappeared. Mutant MCM10 (1-530), MCM10 (1-427), and MCM10 (1-165) were localized in the cytoplasm.  Figure 6A). The MCM10 mutant, 1-655, missing most parts of CTD but still maintaining the MCM2-7 interaction region, was still  Figure 6A). The MCM10 mutant, 1-655, missing most parts of CTD but still maintaining the MCM2-7 interaction region, was still susceptible to degradation by HIV-1, SIVmus, and SIVrcm Vpr. This suggested that the CTD of MCM10 expression may not be affected by being engaged by Vprs. By contrast, the MCM mutant, 1-530, missing both MCM 2-7 interaction and CTD domains, was resistant to degradation by Vpr mediation. Moreover, the truncation mutants, 1-427 and 1-165, were both resistant to downregulation by Vprs. Collectively, the region 530-655 appears to be a key region that is responsible for MCM10 proteolysis by Vpr. In order to confirm above results, MCM10 1-655 or 1-530 was co-transfected with 3 Vprs, following which coherent results were obtained via western blotting ( Figure 6B), where MCM10 1-655 retained susceptibility to Vprs, while MCM 1-530 showed resistance to degradation by Vprs.

MCM10 Failure to Alleviate DDR Inducted by Primate Lentiviruses Vprs
Previous studies showed that MCM10 prevented DNA damage during the replication process [20]. The speculation that DNA damage response (DDR) may be involved in the degradation process led to the need to explore possible consequences of MCM10 degradation by Vprs. Variant histone H2AX (H2AX), a DDR marker, is phosphorylated (γ-H2AX) in response to DNA double strand breaks (DSBs) by intra-or inter-pathogens or other environmental irritants. Firstly, we investigated whether these 3 Vprs provoked a DNA damage response. HEK293 cells were transiently transfected with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus and SIVrcm Vprs, or the control pcDNA3.1/3 × FLAG, following which transfected cells were examined for γ-H2AX foci formation, using immunofluorescence staining with anti-γ-H2AX mAb. As shown, HIV-1, SIVmus, and SIVrcm Vpr induced γ-H2AX foci to aggregate in the nucleus of HEK293 cells ( Figure 7A). By contrast, HEK293 cells transfected with the control vector was negative for immunofluorescence of γ-H2AX. Similarly, In order to confirm above results, MCM10 1-655 or 1-530 was co-transfected with 3 Vprs, following which coherent results were obtained via western blotting ( Figure 6B), where MCM10 1-655 retained susceptibility to Vprs, while MCM 1-530 showed resistance to degradation by Vprs.

MCM10 Failure to Alleviate DDR Inducted by Primate Lentiviruses Vprs
Previous studies showed that MCM10 prevented DNA damage during the replication process [20]. The speculation that DNA damage response (DDR) may be involved in the degradation process led to the need to explore possible consequences of MCM10 degradation by Vprs. Variant histone H2AX (H2AX), a DDR marker, is phosphorylated (γ-H2AX) in response to DNA double strand breaks (DSBs) by intra-or inter-pathogens or other environmental irritants. Firstly, we investigated whether these 3 Vprs provoked a DNA damage response. HEK293 cells were transiently transfected with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus and SIVrcm Vprs, or the control pcDNA3.1/3 × FLAG, following which transfected cells were examined for γ-H2AX foci formation, using immunofluorescence staining with anti-γ-H2AX mAb. As shown, HIV-1, SIVmus, and SIVrcm Vpr induced γ-H2AX foci to aggregate in the nucleus of HEK293 cells ( Figure 7A). By contrast, HEK293 cells transfected with the control vector was negative for immunofluorescence of γ-H2AX. Similarly, γ-H2AX expression in all three groups of HEK293T cells transfected with either pcDNA3.1/3 × FLAG-HIV-1, SIVmus, or SIVrcm Vprs was increased, while γ-H2AX expression in cells transfected with the control pcDNA3.1/3 × FLAG vector was not ( Figure 7B).

Correlation of MCM10 Degradation with HIV-1 Vpr G 2 /M Arrest
Previously, HIV-1 Vpr was found to enhance its G 2 /M arrest effect by increasing MCM10 degradation via proteasome-dependent pathway [18]. However, considering multiple functions of HIV-1 Vpr to cellular targets, a question arose as to whether other functions of Vpr also played roles. Firstly, seven typical HIV-1 Vpr mutants were summed up and characterized ( Figure 8A). HIV-1 Vpr K27M, C76A, and R80A curbed the cell cycle at the G 2 /M phage [15,31,32]; K27M, R77Q, and R80A impaired the function of apoptosis induction [15]; P35A specifically lacked Vpr oligomerization [33]; and W54R failed to interact with the host factor UNG2 [34]. Secondly, we generated the expression vectors pME18neo/FLAG-Vpr-IRESZsGreen1 encoding the HIV-1 Vpr mutants, K27M, P35A, W54R, C76A, R77Q, and R80A, and transfected HEK293T cells for cell cycle analysis. The expression of all Vpr mutants were confirmed by western blotting. Three mutants, K27M, C76A, and R80A, decreased cell cycle arrest activity at the G 2 /M phase compared with wildtype HIV-1 ( Figure 8B), indicating the three mutants failed to induce cell cycle blocking.
Finally, quantitative data related to MCM10 degradation profiles and G 2 /M:G 1 ratios, associated with Vpr mutants, indicated a high correlation (R 2 = 0.8589; p = 0.0009) with each of the functions tested ( Figure 8D). The G 2 /M arrest function of HIV-1 Vpr was specifically correlated with MCM10 degradation. By contrast, R77Q, an apoptosis induction-deficient mutant, downregulated MCM10 expression, demonstrating that MCM10 expression levels were not affected by the apoptosis function of HIV-1 Vpr. Another mutant P35A, which lost the Vpr oligomerization function, also failed to reverse MCM10 degradation.

Correlation of MCM10 Degradation with HIV-1 Vpr G2/M Arrest
Previously, HIV-1 Vpr was found to enhance its G2/M arrest effect by increasing MCM10 degradation via proteasome-dependent pathway [18]. However, considering multiple functions of HIV-1 Vpr to cellular targets, a question arose as to whether other functions of Vpr also played roles. Firstly, seven typical HIV-1 Vpr mutants were summed up and characterized ( Figure 8A). HIV-1 Vpr K27M, C76A, and R80A curbed the cell cycle at the G2/M phage [15,31,32]; K27M, R77Q, and R80A impaired the function of apoptosis induction [15]; P35A specifically lacked Vpr oligomerization [33]; and W54R failed to interact with the host factor UNG2 [34]. Secondly, we generated the expression vectors pME18neo/FLAG-Vpr-IRESZsGreen1 encoding the HIV-1 Vpr mutants, K27M, P35A, W54R, C76A, R77Q, and R80A, and transfected HEK293T cells for cell cycle analysis. The expression of all Vpr mutants were confirmed by western blotting. Three mutants, K27M, C76A, and R80A, decreased cell cycle arrest activity at the G2/M phase compared with wildtype HIV-1 ( Figure 8B), indicating the three mutants failed to induce cell cycle blocking.
Finally, quantitative data related to MCM10 degradation profiles and G2/M:G1 ratios, associated with Vpr mutants, indicated a high correlation (R 2 = 0.8589; p = 0.0009) with each of the functions tested ( Figure 8D). The G2/M arrest function of HIV-1 Vpr was specifically correlated with MCM10 degradation. By contrast, R77Q, an apoptosis induction-deficient mutant, downregulated MCM10 expression, demonstrating that MCM10 expression levels were not affected by the apoptosis function of HIV-1 Vpr. Another mutant P35A, which lost the Vpr oligomerization function, also failed to reverse MCM10 degradation.

Discussion
Previous studies have indicated that HIV-1 Vpr increased MCM10 degradation by manipulating DCAF1-Cul4-E3 ubiquitin ligase for proteasome dependent degradation pathway and that such degradation was related to Vpr-mediated G2/M arrest. However, it was unclear whether various primate lentiviruses Vprs also complied with MCM10 proteasome dependent degradation. The current study reached three major conclusions regarding MCM10 degradation pattern by various primate lentiviruses Vpr proteins. Firstly, the study revealed that MCM10 degradation resulting from identical proteasome pathways was caused by distinct SIVmus and SIVrcm Vprs, in addition to HIV-1 Vpr. However, Vpr proteins derived from prototype virus lineages lost the ability to degrade Sources: [5,7,11,13,15,[31][32][33][34][35][36][37]. (B) HEK293 cells were transfected with pME18neo/FLAG-IRESZsGreen1 that encoded FLAG-tagged HIV-1 Vpr wild type and a panel of mutants stated above. At 48 h after transfection, cells were harvested to analyze DNA content and stained with propidium iodide. ZsGreen1-positive cells were analyzed using a BD Accuri TM C6 Plus with a sampler flow cytometer. For each mutant, 10000 events were acquired and subsequent G 2 /M:G 1 ratio was calculated using FlowJo software. (C) HEK293T cells were transiently transfected with either pcDNA3.1/HA-MCM10 together with either HIV-1 pME18neo FLAG-tagged HIV-1 Vpr, wild type and a panel of mutants. Transfected cells were harvested at 48 h after transfection and lysates with the equal protein amounts were subjected to western blotting (left panel). The positions of 3 × FLAG-Vpr, MCM10 and α-Tubulin are indicated. Band densities of HA-MCM10 and α-Tubulin were analyzed by densitometry analysis using ImageJ software (right panel). The relative intensities were calculated as the ratio of density of MCM10 to density of α-Tubulin. Each column and error bar represents the mean ± SD for three independent experiments. The asterisks indicate a statistically significant differences (** p < 0.01, *** p < 0.001). (D) Correlation between MCM10 degradation and G 2 /M arrest by HIV-1 Vpr mutants. The line represents the approximate curve. R = Pearson's correlation coefficient (p = 0.0009).

Discussion
Previous studies have indicated that HIV-1 Vpr increased MCM10 degradation by manipulating DCAF1-Cul4-E3 ubiquitin ligase for proteasome dependent degradation pathway and that such degradation was related to Vpr-mediated G2/M arrest. However, it was unclear whether various primate lentiviruses Vprs also complied with MCM10 proteasome dependent degradation. The current study reached three major conclusions regarding MCM10 degradation pattern by various primate lentiviruses Vpr proteins. Firstly, the study revealed that MCM10 degradation resulting from identical proteasome pathways was caused by distinct SIVmus and SIVrcm Vprs, in addition to HIV-1 Vpr. However, Vpr proteins derived from prototype virus lineages lost the ability to degrade MCM10, implying that MCM10 degradation is associated with species specificity of Vprs. Secondly, our results demonstrated that MCM10 interacted with HIV-1, SIVmus, and SIVrcm Vprs. Furthermore, the MCM2-7 interaction region of MCM10 was the determinant region susceptible to degradation by these 3 Vprs. Thirdly, our data showed that although the γ-H2AX expression levels increased, the 3 Vprs remained unaltered following overexpression of MCM10 in 293T cells, suggesting that MCM10 did not alleviate the DNA damage response induced by the 3 Vprs.
In this study, according to phylogenetic outcomes, 10 representative Vpr proteins from different primate lentiviruses lineages were selected and synthesized. Interestingly, a potential zinc-binding motif (H33, H71, H76, and *78) [38] was found among α-helices 2 and 3 in the Vpr proteins from diverse virus strains. Multiple zinc-binding regions involved in viral proteins are indispensable for negotiating with host factors. For instance, the zinc-binding region (HX 5 CX 17-18 CX 3-5 H) of HIV-1 Vif mediated interaction with Cul5 E3 ligase to exert ubiquitination targeting APOBEC3G [39,40]. Two other zinc-binding sites in the nucleocapsid (NC) of HIV-1 also play an important role in the interaction with nucleus acids of PSI RNA and the eventual promotion of HIV-1 genomic RNA packaging into virus particles [41,42]. However, there is little evidence indicating whether such Vpr sequences mimic the full role played by typical HHCC zinc-binding motifs. Recently, some studies have revealed that the HHCH motif of HIV-1 Vpr, which is positioned parallel to that of HIV-2 Vpx, may show capacity to interact with the E3 ligase complex [28]. However, whether the potential zinc-binding motif contributes to the function of Vprs remains unclear.
Among Vprs from 10 lineages, HIV-1, SIVmus, and SIVrcm Vprs were identified as having varying capacity to specifically curb the expression of MCM10, while other strains failed to do so. Furthermore, MCM10 was colocalized with such Vprs in the nucleus and formed complexes with them. Interestingly, it is suggested that Vprs originating from prototype viruses are unable to induce downregulation of MCM10. Multiple alignment results indicated that there were no distinguishable sequences or point features that would enable differentiation of the capacity to degrade MCM10. Accordingly, it is speculated that more than one amino acid, or a combination of amino acids, in distinct virus strains may perform the function of MCM10 degradation. However, more proof is required to determine whether Vpr proteins of whole prototype virus lineages exhibit MCM10 degradation properties.
The region encompassing amino acids 530-655, also known as the MCM2-7 interaction region, was mapped as a determinant domain of MCM10 degradation under induction by Vpr [20,43]. Previously, little was known about the 530-633 region of MCM10, identified as a newly identified functional domain, flanked by an ID and involved in parts of CTD. This region exhibits little sequence characterization or secondary structures, by way of sequence alignments and structure prediction, in spite of the compositional bias of hydrophobic amino acids [44,45]. This flexible region is also partly responsible for the intrinsically disordered proteins (IDP). Some studies revealed that IDPs adopted multiple structures and were inclined to enfold, thus mediating binding with other targets of interest [46].
Once released into target cells, a virion-associated HIV-1 Vpr initiates multiple functions to facilitate its replication. MCM10 degradation by HIV-1 Vpr was found to induce G 2 /M cell arrest in Hela cells [18]. However, whether Vpr played other roles in MCM10 degradation remains unknown. Therefore, we constructed a series of functionally deficient Vpr mutants and performed MCM10 degradation profiling. Our mutagenesis assay indicated that the G 2 /M cell cycle, instead of apoptosis induction, oligomerization, or nuclear localization, was correlated with MCM10 expression. This finding supported the key role played by MCM10 in cell cycle modulation.
Diverse viruses negotiate with and eliminate cellular factors, by exploiting host metabolism pathways to facilitate virus replication and escape host immune surveillance. Particularly, the ubiquitin dependent degradation pathway is one of the attractive machineries manipulated by multiple accessory proteins, such as, Vif, Vpu, and Vpx, of primate lentiviruses. Predominantly, Vpr was found to invoke increasingly numerous host factors for proteasome dependent degradation, such as MCM10, MUS81, helicase-like transcription factor (HLTF), Exonuclease 1 (Exo1), and histone deacetylases (HDACs) [35][36][37]47,48]. Interestingly, these cellular targets also respond to DNA damage from viruses or other environmental stimulants. For instance, HLTF labels the proliferating cell nuclear antigen (PCNA) with Lys-63 polyubiquitin chain to reverse leading strand replication with that of the lagging strand, a rather undamaged template at replication level error correction. Exo1, another target of HIV-1 Vpr, was also depleted via the proteasomal degradation pathway. Vpr may possibly load Exo1 onto the E3 ligase complex and remodel the post-replication DNA repair machinery independently of PCNA bridging. However, the correlation between Exo1 depletion by Vpr and DNA damage response remains unknown [48,49].
DNA damage and late S/G2 phase arrest are induced through MCM10 siRNA treated cells. During replication, MCM10 depletion supposedly blocks the synthesis of the lagging DNA strand, and the subsequent replication fork stalling also generates phosphorylated H2AX. The DSB signal cascade eventually leads to cell cycle arrest [50]. In addition, HIV-1 was found to enhance MCM10 degradation which invoked G 2 /M cell cycle blocking [18]. Mutagenesis and cell cycle analysis also revealed that the degree of MCM10 degradation was correlated with cell cycle arrest with HIV-1 Vpr. However, in our study, MCM10 alone does not alleviate the DNA damage induced by 3 Vpr proteins, suggesting complexity of DNA modulation exposing to viral pathogens. It is still unclear whether the primary role of Vpr on DNA damage exceeds resultant DNA repair machinery activation to favor the purpose of virus replication. Taken together, the results of our studies highlight distinct interplay model of host factor MCM10 with various primate lentiviruses Vpr proteins and their ensuing roles on physiological alternation of cellular targets partly. This research presents a model of primate lentiviruses Vprs antagonism against increasingly found host factors and "arm-race" of host-virus coevolution.

Conclusions
This study revealed that distinct MCM10 degradation profiles by primate lentiviruses Vpr/Vpx proteins through proteasome degradation pathway. Particularly, HIV-1, SVImus and SIVrcm Vpr curbed MCM10 expression, while Vpr derived from other 8 Vpr/Vpx did not. Interestingly, colocalization and interaction of MCM10 and these three Vpr proteins also were observed. And MCM2-7 interaction region was susceptible to degradation through proteasomal degradation pathway. For HIV-1, G2/M interruption was directly related with MCM10 degradation but other Vpr function defects were not. However, further experiment is need to investigate whether such set of DNA damage response proteins exert synergistic roles to interact with accessory protein Vpr. Funding: This study was partly supported by a grant to YA (Research on HIV/AIDS project no. H26-002) from the Ministry of Health, Labor, and Welfare of Japan, and by grants to YA (Research on HIV/AIDS project nos. 18fk0410004h0203, 17fk0410204h0402, 16fk0410104h0501 and 16fk0410302h0003) from the Japan Agency for Medical Research and Development.