UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation

Abstract

Over 50% of the world’s population is infected with Human Cytomegalovirus (HCMV). HCMV is responsible for serious complications in the immuno-compromised and is a leading cause of congenital birth defects. The molecular function of many HCMV proteins remains unknown, and a deeper understanding of the viral effectors that modulate virion maturation is required. In this study, we observed that UL34 is a viral protein expressed with leaky late kinetics that localises to the nucleus during infection. Deletion of UL34 from the HCMV genome (ΔUL34) did not abolish the spread of HCMV. Instead, over >100-fold fewer infectious virions were produced, so we report that UL34 is an augmenting gene. We found that ΔUL34 is dispensable for viral DNA replication, and its absence did not alter the expression of IE1, MCP, gB, UL26, UL83, or UL99 proteins. In addition, ΔUL34 infections were able to progress through the replication cycle to form a viral assembly compartment; however, virion maturation in the cytoplasm was abrogated. Further examination of the nucleus in ΔUL34 infections revealed replication compartments with aberrant morphology, containing significantly less assembled capsids, with almost none undergoing subsequent maturation. Therefore, this work lays the foundation for UL34 to be further investigated in the context of nuclear organization and capsid maturation during HCMV infection.

Keywords: HCMV; UL34; capsid maturation; genome packaging; herpesvirus; replication compartment.

Figure 1

RNA sequencing and bioinformatics analysis of HCMV genes. (A) Heatmap depicting HCMV gene features based on complete linkage clustering and Gower distance. Columns represent the relative log₂ fold-change in transcript abundance in ΔUL54 mutant infections compared to WT at 72 HPI, relative log₂ virion enrichment compared to a WT infected cellular lysate at 5 DPI [36] (positive values reflect virion enrichment), kinetic expression classes 1 to 5 based on Weekes et al. [19] and essential/augmenting/non-essential gene classification based on Yu et al. [37]. (B) Heatmap depicting HCMV genes expanded from clusters I and II in (A), and sub-clustered based on complete linkage clustering and Gower distance. (C) t-stochastic neighbour embedding plot of genes from (B).

Figure 2

Characterisation of UL34 as an augmenting viral protein with leaky late expression kinetics. (A) Intracellular HCMV genome copies at 120 HPI, relative to genome copies at 12 HPI. MOI = 3, n = 3, bars = SD. (B) Western blot analysis of UL34 expression in MRC5 cells treated with 100 μg/mL phosphonoacetic acid (PAA) and subsequently infected with either AD169-GFP WT or AD169 HA-UL34 HCMV (5 DPI, MOI = 3). Membranes were probed with primary antibodies against HCMV viral proteins, HA, or β-actin loading control. (C) Immuno-fluorescence analysis of host GM130 and viral UL99 in WT MRC5 cells infected with WT or ΔUL34 AD169-GFP virus. 4 DPI, MOI = 0.1, scale bars = 20 μm. (D) Growth kinetics of ΔUL34 AD169-GFP virus, as measured by IE1 fluorescent focus assay in cell culture supernatants from WT and UL34-complementing MRC5 cells. MOI = 3, n = 3, bars = SD. (E) Spread of ΔUL34 AD169-GFP virus in WT and UL34-complementing MRC5 cells, as quantified by fixing, staining, and counting IE1 positive cells at indicated time points. MOI = 0.01, n = 3, bars = SD.

Figure 3

Characterisation of UL34 as a nuclear viral protein dispensable for viral gene expression. (A) Western blot analysis of UL34 expression kinetics. MRC5 cells were infected with HCMV AD169 HA-UL34 (endogenous n-terminal HA tag and linker) virus and lysed at various times post-infection. Mock and WT AD169-GFP WT control conditions were lysed at 5 DPI (MOI = 3). Membranes were probed with primary antibodies against HCMV viral proteins, HA, or β-actin loading control. (B) Immuno-fluorescence analysis of MRC5 cells infected with AD169-GFP or AD169 HA-UL34 HCMV and stained with HA (UL34) and GM130 antibodies. 4 DPI, MOI = 0.1, scale bars = 20 μm. (C) Western blot analysis of lysates from MRC5 and UL34 complementing cells infected with either WT or ΔUL34 HCMV AD169-GFP virus. 5 DPI, MOI = 3. Membranes were probed with primary antibodies against HCMV viral proteins or β-actin loading control.

Figure 4

Analysis of UL34 protein interactions. Raw spectral files from UL34 immunoprecipitation (IP) experiments generated by Nobre et al. [56] were downloaded and researched using MaxQuant. (A) Volcano plot showing relative enrichment of proteins in the UL34 IP (positive fold change), compared to control. Purple: UL34 bait, Blue: cadherins, Green: nuclear lamins, Pink: chromatin modifiers, Red: PP4 phosphate complex sub-units, Black circles: significant differential expression. n = 2, S₀ = 2, FDR < 0.05. (B) Bubble plot depicting significantly enriched Gene Ontology terms from the list of UL34 interactors, compared to background (p < 10⁻³). The x-axis represents the fold enrichment of the term in the target list compared to the expected number based on the background. Bubble size represents the number of IDs associated with each term. (C) Immuno-fluorescence analysis of GM130 and lamin A/C in WT MRC5 cells infected with WT or ΔUL34 AD169-GFP virus. 4 DPI, MOI = 0.1, scale bars = 20 μm. (D) Immuno-fluorescence analysis of GM130 and lamin B1 in WT MRC5 cells infected with WT or ΔUL34 AD169-GFP virus. 4 DPI, MOI = 0.1, scale bars = 20 μm.

Figure 5

TEM analysis of cells infected with WT HCMV or ΔUL34 virus. (A) Representative electron micrograph of an MRC5 cell infected with WT AD169-GFP virus. 5 DPI, MOI = 1, scale bar = 10 μm. (B,C) Inset areas from (A) show the cytoplasmic vAC and nucleus, respectively. Scale bars = 2 μm. (D) Representative electron micrograph of an MRC5 cell infected with ΔUL34 AD169-GFP virus. 5 DPI, MOI = 1, scale bar = 10 μm. (E,F) Inset areas from (D) show the cytoplasmic vAC and nucleus, respectively. Scale bars = 2 μm. (G,H) Representative electron micrographs depicting the distinct capsid types in the nuclei of MRC5 cells infected with WT or ΔUL34 AD169-GFP virus, respectively. White arrowhead: A capsids, Black arrowhead: B capsids, Black arrow: C capsids, 5 DPI, MOI = 1, scale bars = 500 nm. (I) Bar chart depicting the total number of each capsid type (A, B or C) in WT infected nuclei cross-sections. Bars = SD, n = 10. (J) Bar chart depicting the total number of each capsid type in ΔUL34 infected nuclei cross-sections. Bars = SD, n = 12. (K–M) Percentage of B, A, and C capsids in WT or ΔUL34 infected nuclei cross-sections shown in (I,J). WT n = 10, ΔUL34 n = 12, bars = SD, * p < 0.05, *** p < 0.001, two-tailed t-test with Welch’s correction.