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Human immunodeficiency virus type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest.

Sakai K, Barnitz RA, Chaigne-Delalande B, Bidère N, Lenardo MJ - Virol. J. (2011)

Bottom Line: To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition.We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif.Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation.

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Vif-induced cell cycle arrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3e-n-GFP e-f+r+ (A and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP+ cells was examined by flow cytometry using DRAQ5 at 24 and 42 hours post-infection as previously described [4]. The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cell cycle model and the ratio was plotted [4]. All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55,57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54-56], and mouse-anti-β-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.
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Figure 4: Vif-induced cell cycle arrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3e-n-GFP e-f+r+ (A and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP+ cells was examined by flow cytometry using DRAQ5 at 24 and 42 hours post-infection as previously described [4]. The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cell cycle model and the ratio was plotted [4]. All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55,57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54-56], and mouse-anti-β-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.

Mentions: A recent study reported that Vif induced a delay in cell cycle rather than complete arrest [15]. It is difficult to compare our results and those reported by DeHart and colleagues because the experimental system used in their study, especially the cells (SupT1 cells) and level of infection (multiplicities of infection [MOI] of 1-2), is different from ours [3,15]. We observed that the G2 arrest due to Vif alone was most pronounced at a high MOI. Furthermore, unlike the combination of Vpr and Vif, Vif-induced cell cycle arrest showed a direct relationship with increasing MOI (and therefore the increasing expression level of Vif), whereas the arrest caused by the e-f+r+ virus appeared to be independent of MOI (Figure 4A-D). However, similar to the cell cycle blockade caused by the e-f+r+ virus, cells infected with the e-f+r- virus showed the highest G2/G1 ratio on day 2 post-infection, when the expression of the provirus peaks (Figure 4 and data not shown). We observed strong cell cycle arrest caused by high levels of Vif expression in both synchronized and non-synchronized Jurkat cells (Figures 1B and 4B). As previously shown [3], the e-f-r- virus caused no significant G2 arrest (Figure 4E and 4F). Thus, high expression of Vif arrested cells in the G2 phase, although not to the same degree as the combined expression of Vpr and Vif.


Human immunodeficiency virus type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest.

Sakai K, Barnitz RA, Chaigne-Delalande B, Bidère N, Lenardo MJ - Virol. J. (2011)

Vif-induced cell cycle arrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3e-n-GFP e-f+r+ (A and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP+ cells was examined by flow cytometry using DRAQ5 at 24 and 42 hours post-infection as previously described [4]. The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cell cycle model and the ratio was plotted [4]. All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55,57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54-56], and mouse-anti-β-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Vif-induced cell cycle arrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3e-n-GFP e-f+r+ (A and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP+ cells was examined by flow cytometry using DRAQ5 at 24 and 42 hours post-infection as previously described [4]. The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cell cycle model and the ratio was plotted [4]. All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55,57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54-56], and mouse-anti-β-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.
Mentions: A recent study reported that Vif induced a delay in cell cycle rather than complete arrest [15]. It is difficult to compare our results and those reported by DeHart and colleagues because the experimental system used in their study, especially the cells (SupT1 cells) and level of infection (multiplicities of infection [MOI] of 1-2), is different from ours [3,15]. We observed that the G2 arrest due to Vif alone was most pronounced at a high MOI. Furthermore, unlike the combination of Vpr and Vif, Vif-induced cell cycle arrest showed a direct relationship with increasing MOI (and therefore the increasing expression level of Vif), whereas the arrest caused by the e-f+r+ virus appeared to be independent of MOI (Figure 4A-D). However, similar to the cell cycle blockade caused by the e-f+r+ virus, cells infected with the e-f+r- virus showed the highest G2/G1 ratio on day 2 post-infection, when the expression of the provirus peaks (Figure 4 and data not shown). We observed strong cell cycle arrest caused by high levels of Vif expression in both synchronized and non-synchronized Jurkat cells (Figures 1B and 4B). As previously shown [3], the e-f-r- virus caused no significant G2 arrest (Figure 4E and 4F). Thus, high expression of Vif arrested cells in the G2 phase, although not to the same degree as the combined expression of Vpr and Vif.

Bottom Line: To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition.We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif.Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation.

Show MeSH
Related in: MedlinePlus