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. 2023 Aug 31;97(8):e0078123.
doi: 10.1128/jvi.00781-23. Epub 2023 Aug 11.

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

Elisa Fanunza  1   2 , Adam Z Cheng  3 , Ashley A Auerbach  1 , Bojana Stefanovska  1   4 , Sofia N Moraes  3 , James R Lokensgard  5 , Matteo Biolatti  6 , Valentina Dell'Oste  6 , Craig J Bierle  7 , Wade A Bresnahan  8 , Reuben S Harris  1   4
Affiliations

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

Elisa Fanunza et al. J Virol. .

Abstract

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

Keywords: APOBEC3B (A3B); herpesviruses; human cytomegalovirus; immediate-early genes; innate immunity; ribonucleotide reductase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
A3B relocalization occurs with multiple HCMV strains in different cell types. (A–E) Representative IF microscopy images of the indicated cell types stably expressing A3B-HA incubated with medium alone (mock) or infected with the indicated HCMV strains for 72 h (10 µm scale). High-magnification images in Fig. 1A were taken with a Nikon Eclipse Ti2 system, and all other images were taken with an EVOS cell imaging system. (F and G) Quantification of A3B-HA subcellular localization phenotypes shown in panels A–E. Each histogram bar reports the percentage of cells with cytoplasmic A3B-HA (n > 100 cells per condition; mean ± SD with indicated P values from unpaired Student’s t-tests).
Fig 2
Fig 2
Catalytic mutant and endogenous A3B are relocalized by HCMV. (A–E) Representative IF microscopy images of the indicated cell types stably expressing A3B-E255A-HA incubated with medium alone (mock) or infected with the indicated HCMV strains for 72 h (10 µm scale). (F) Quantification of A3B-E255A-HA subcellular localization phenotypes shown in panels A–E. Each histogram bar reports the percentage of cells with cytoplasmic A3B-HA (n > 100 cells per condition; mean ± SD with indicated P values from unpaired Student’s t-tests). (G) Representative IF microscopy images of ARPE19 cells incubated with medium alone (mock) or infected with TB40-mCherry for 72 h and then stained for endogenous A3B (10 µm scale). (H) Quantification of endogenous A3B subcellular localization phenotype shown in panel G. The dot-plot chart shows the ratio between nuclear and cytoplasmic fluorescence intensity (n > 50 cells per condition; P values obtained with unpaired Student’s t-tests).
Fig 3
Fig 3
A3B relocalization is UL45 independent. (A) Co-IP of transfected HCMV UL45-FLAG with the indicated A3-HA constructs in 293T cells. Cells co-transfected with EBV BORF2 and A3B or A3G are used as positive and negative controls, respectively. (B) TBE-urea PAGE analysis of A3B deaminase activity in the presence of empty vector, HCMV UL45, or EBV BORF2. (C) Representative IF microscopy images of HeLa cells transiently expressing A3B-HA together with empty vector, HCMV UL45-FLAG, or EBV BORF2-FLAG (10 µm scale). (D and E) Representative IF microscopy images of the indicated cell types stably expressing A3B-HA incubated with medium alone (mock) or infected with the indicated HCMV strains and UL45-null derivatives for 72 h (10 µm scale).
Fig 4
Fig 4
The NTD of A3B is sufficient for A3B relocalization mediated by HCMV. (A) Representative IF microscopy images of ARPE19 cells transiently expressing EGFP alone, A3B-FL-EGFP, A3B-NTD-EGFP, and A3B-CTD-EGFP, incubated with medium alone (mock) or infected with TB40-mCherry for 72 h (10 µm scale). (B) Quantification of A3B-FL, A3B-NTD, and A3B-CTD subcellular localization phenotype shown in panel A. The dot-plot chart shows the ratio between nuclear and cytoplasmic fluorescence intensity (n > 25 cells per condition; P values obtained with unpaired Student’s t-tests). (C) Representative IF microscopy images of HeLa cells transiently expressing EBV BORF2-FLAG together with EGFP alone, A3B-FL-EGFP, A3B-NTD-EGFP, and A3B-CTD-EGFP (10 µm scale).
Fig 5
Fig 5
A3B relocalization occurs early during HCMV infection. (A and C) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with the indicated HCMV strains for the indicated timepoints (10 µm scale). (B and D) Quantification of A3B-HA subcellular localization phenotypes shown in panels A and C. Each histogram bar reports the percentage of cells with whole cell, cytoplasmic, and nuclear A3B-HA (n > 100 cells per condition; mean ± SD with P values from unpaired Student’s t-tests).
Fig 6
Fig 6
A3B relocalization requires de novo translation of HCMV proteins but not viral DNA synthesis. (A) Schematic representation of experimental workflows of CHX and PAA treatment of infected cells. Image created with BioRender. (B) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP and treated with DMSO or CHX for 24 h (10 µm scale). (C) Representative IF microscopy images of U373 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP or AD169-GFP ΔIE1 for 72 h (10 µm scale). (D) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP and treated with DMSO or PAA for 48 h (10 µm scale). (E) Quantification of A3B-HA subcellular localization phenotypes shown in panels B and D. Each histogram bar reports the percentage of cells with cytoplasmic A3B-HA (n > 80 cells per condition; mean ± SD with P values from unpaired Student’s t-tests).
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