Vif and Vpr induce distinct forms of mitotic arrest.

(A) Representative live cell image for Cal51 cells with H2B-mScarlet and Tubulin-mNeonGreen expressing Control, Vif, Vpr, or Vif+Vpr reporter viruses. (B) Average mitotic duration of Cal51 cells expressing respective reporter virus (n=100 for each from two replicates). (C) Frequency of cell fate after mitosis for Cal51 cells expressing respective reporter virus. (n=100 for each from two replicates)

Vif induces robust pseudo-metaphase arrest.

(A) Representative live cell images for Cal51 cells with H2B-mScarlet and Tubulin-mNeonGreen expressing Control reporter virus. (B) Representative live cell image for Cal51 cells expressing Vif reporter virus. (C) Frequency of cells that achieve metaphase plate and time taken to achieve metaphase plate for cells in (A) and (B) (n=100 for each from two replicates). (D) Representative live-cell images of Vif conditional expressed HeLa cell with or without Doxycycline. (E) Average mitotic duration in condition (D) (n=100 cells for each from two replicates). (F) Quantification of viable cells over time after Dox induction. (G) Quantification of apoptotic cells after Dox induction.

Vif does not alter G1 or S phase progression, accelerates G2 progression, and induces pseudo-metaphase arrest independent of p53

(A) Top: Representative image of Cal51 cells progressing through G1, S, and G2. Bottom: Representative trace for relative signal intensity of the nucleus through cell cycle. (B) Average duration of G1, S, and G2 phases in Control and Vif-expressing cells (n= 9 for Control and 11 for Vif, from two replicates). (C) Total cell cycle duration for Control and Vif-expressing cells. (D) Representative live cell images for Control and Vif-expressing WT or p35KO RPE1 cells. (E) Average mitotic duration in WT or p53KO RPE1 cells (n= >85 cells, from two replicates). (F) Frequency of cells which achieve metaphase plate for cells in (E). (G) Average time taken to achieve metaphase plate for cells in (F).

Vif induces polar chromosomes, multi-polar spindles, and abnormal chromosome movements

(A) Representative immunofluorescence images labeled for CENP-C (as a kinetochore marker), microtubule, and DNA in Control and Vif-expressing HeLa cells. (B) Example super-resolution images labeled for CENP-C, microtubule, and DNA in Vif-expressed HeLa cells showing polar chromosomes. (C) Representative live cell image of Vif-expressing cells where polar chromosomes were quantified by compartmentalizing polar regions. Bottom: Quantification of polar chromosome frequency overtime. (D) Representative high-temporal live cell images (6 minutes interval) showing rapid chromosome moving towards and away from the spindle poles. (E) Fraction of Cal51 cells showing abnormal number of poles at some point during mitosis. (F) Top: Representative images of maximum mitotic spindle length for Control and Vif-expressing Cal51 cells. Bottom: Average maximum mitotic spindle length of Control and Vif-expressing cells. (G) Top: Representative live cell image of Control and Vif-expressing Cal51 cells over time showing dynamic spindle spinning. Center: Representative figures showing relative orientation (angle) of the spindle axis over time (radius). Bottom: Average total angle swept during mitosis.

Vif, but not Vpr, disrupts the proper localization of PP2A-B56 at the kinetochores

(A) Representative maximum intensity projection of fixed Control and Vif-expressing HeLa cells with or without nocodazole treatment, immunofluorescently labeled for B56, CENP-C, and DNA. (B) Representative maximum intensity projection of fixed Control and Vpr-expressing HeLa cells, immunofluorescently labeled for B56, CENP-C, and DNA. (C) Average B56 level at kinetochores for cells in (A) and (B) (n=200 kinetochores over 8 cells from two independent replicates for each). (D) Representative fixed-cell immunofluorescent image of HeLa cells expressing Vif, immunofluorescently labeled for Plk1, CENP-C, and DNA. (E) Average Plk1 level at kinetochores for cells in (D) (n=200 kinetochores over 8 cells from two independent replicates for each).

Vif impairs stable and balanced kinetochore microtubule attachments

(A) Left: Representative maximum intensity projection of fixed Control and Vif-expressing HeLa cells immunofluorescently labeled for CENP-C, Astrin, and DNA along with representative sister kinetochore pairs, Right: Illustrative interpretation of images on the left. (B) Average Astrin and CENP-C level at kinetochores for cells in (A) (n = 200 kinetochores from 8 cells from two independent replicates for each) (C) Comparison of Astrin and CENP-C on sister kinetochores, values normalized with formula: 1 – (lower intensity value/higher intensity value). (D) Representative image of fixed Control and Vif-expressing HeLa cells immunofluorescently labeled for CENP-C, pHec1(S55), and DNA along with representative sister kinetochore pairs. (E) Average pHec1(S55) level at kinetochores for cells in (D). (n = 200 kinetochores from 8 cells from two independent replicates for each). (F) Comparison of pHec1(S55) on sister kinetochores, values normalized with formula: 1 – (lower intensity value/higher intensity value). (G) Representative fixed-cell immunofluorescent image of HeLa cells expressing Vif, immunofluorescently labeled for Hec1, CENP-C, and DNA. (H) Average Hec1 level at kinetochores for cells in (G) (n=200 kinetochores over 8 cells from two independent replicates for each).

Proposed model for the molecular mechanism underlying Vif’s pseudo-metaphase arrest

Top: Cartoon model depicting metaphase alignment of Control cells followed by anaphase. Middle: Cartoon model depicting pseudo-metaphase alignment of Vif-expressing cells with unbalanced microtubule attachment followed by three-step polar chromosome cycle. Bottom: Cartoon depiction of three-step polar chromosome cycle, (1) chromosome at the equator is pulled towards pole due to unbalanced pulling force, (2) kinetochore-microtubule destabilization at the spindle pole, (3) equator-directed movement of chromosome for realignment.

Vif induced a pseudo-metaphase arrest in Cal51 and HeLa cells.

(A) Various HIV-1 NL4-3 reporter viruses without viral Env and Nef, and with CFP used for most experiments in this study. (B) Average mitotic duration for Control and Vif-expressing Cal51 cells, dividing and non-dividing cells shown separately. (C) Representative live cell images of Control and Vif-expressing HeLa cells. (D) Average mitotic duration for Control and Vif-expressing HeLa cells (n=100). (E) Population frequency of cell fate after mitosis. (F) Frequency of cells which achieve metaphase plate for cells in (D). (G) Average time taken to achieve metaphase plate from NEBD for cells in (F). (H) Representative IF images stained for DNA, Tubulin, and CFP in Control and Vif-expressing HeLa cells at 72 hours post-infection (left) and mitotic index (right). (n > 500 cells for each replicate).

Vif induces pseudo-metaphase arrest in MDA-MB-231 cells.

(A) Representative live cell images of Control and Vif-expressing MDA-MB-231 cells. (B) Average mitotic duration for Control and Vif-expressing MDA-MB-231 cells (n=100 from two replicates). (C) Population frequency of cell fate after mitosis. (D) Frequency of cells which achieve metaphase plate during mitosis for cells in (B). (E) Average time taken to achieve metaphase plate from NEBD for cells in (D).

Vif specifically induces pseudo-metaphase arrest independent of p53 status.

(A) Average cell cycle progression from G1 to G2 phase in Cal51 cells for Control and Vif-expressing cells. (B) Duration between consecutive mitosis for RPE1 and MDA-MB-231 cells (C) Population frequency of cell fate after mitosis for WT and p53KO RPE1 cells. (D) Representative live cell images for Control and Vif-expressing WT and p53KO HCT116 cells. (E) Average mitotic duration WT and p53KO HCT116 cells. (n= >70cells). (F) Frequency of cells which achieve metaphase for cells in (E). (G) Average time taken to achieve metaphase plate from NEBD for cells in (F). (H) Population frequency of cell fate after mitosis for WT and p53KO HCT116 cells.

Vif induces unaligned chromosomes, multi-polar spindle, and abnormal chromosome and spindle movements.

(A) Frequency of cells showing at least one mitotic error at 48 hours after doxycycline induced Vif expression in HeLa cells. (B) Population frequency of Control and Vif-expressing cells of various cell lines with polar chromosomes measured with timelapse imaging. (C) Representative fixed cell image of Control and Vif-expressing HeLa cell showing normal vs multipolar spindle along with quantification. (D) Representative plots of individual Cal51 cells showing variable pole number over time. (E) Representative live cell image of Cal51 cells with measured spindle length. (F) Representative plots showing relative position (angle) of spindle axis overtime (radius) for Control and Vif-expressing Cal51 cells.