Extended Data Fig. 9: Host factors that control nuclear polarization are required for efficient HCMV replication.

a, b, siRNAs targeting ATAT1, BICD2, SUN1 or Emerin do not affect the accumulation of viral proteins from different kinetic classes, but reduce the production of infectious virus in cells infected at MOI 1. Reductions in viral yields are similar to reductions in DNA fluorescence intensities detected in Fig. 4c. This suggests that nuclear polarization does not regulate viral gene expression, but maximizes viral DNA replication and production of infectious virus. Bars represent mean ± SEM from 3 independent biological replicates, statistics use two-tailed Student’s t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. c, Depletion of ATAT1, BICD2 or SUN1 potently suppresses HCMV spread. Quantification of plaque areas is shown to the right in e; Bars represent mean ± SEM, statistics use two-tailed Student’s t-test, n = 31-81 plaques total from 3 independent biological replicates, *P ≤ 0.05, **P ≤ 0.01. This data suggests that while viral DNA replication and virus yields are reduced by 50% in a single round of infection, this has cumulative effects on the ability of HCMV to spread to other cells. d–f, General inhibition of Arp2/3 activity has broader effects on HCMV infection. d, The Arp2/3 inhibitor CK-666 suppresses viral gDNA polarization and accumulation. Representative images are shown of viral gene expression. gDNA and its localization relative to H3K9me3 foci. e, HCMV spread is suppressed in CK-666-treated, but not inactive control CK-689-treated cells. Plaque areas are shown, bars represent mean ± SEM, statistics use two-tailed Student’s t-test, n = 416 plaques total from 3 independent biological replicates, *P ≤ 0.05, **P ≤ 0.01. f, Arp2/3 inhibition suppresses viral protein accumulation and virus yields in single cycle infections. These data show that inhibition of actin polymerization using Arp2/3 inhibitors not only blocks nuclear polarization but has additional effects on viral gene expression, including modest effects on IE2 expression and more noticeable effects on intermediate and late proteins. This suggests roles for both nuclear and cytoplasmic actin when using broad-spectrum inhibitors over more targeted approaches against nuclear actin alone. Bars represent mean ± SEM from 3 independent biological replicates, statistics use two-tailed Student’s t-test, *P ≤ 0.05, ***P ≤ 0.001. For all experiments, data shown is representative of 3 independent biological replicates.