Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity.

The apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-ß), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-ß by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCE APOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-ß. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING.

The innate immune response plays a pivotal role during human cytomegalovirus (HCMV) primary infection. Indeed, HCMV infection of primary fibroblasts rapidly triggers strong induction of type I interferons (IFN-I), accompanied by proinflammatory cytokine release. Here, we show that primary human foreskin fibroblasts (HFFs) infected with a mutant HCMV TB40/E strain unable to express UL83-encoded pp65 (v65Stop) produce significantly higher IFN-ß levels than HFFs infected with the wild-type TB40/E strain or the pp65 revertant (v65Rev), suggesting that the tegument protein pp65 may dampen IFN-ß production. To clarify the mechanisms through which pp65 inhibits IFN-ß production, we analyzed the activation of the cGAS/STING/IRF3 axis in HFFs infected with either the wild type, the revertant v65Rev, or the pp65-deficient mutant v65Stop. We found that pp65 selectively binds to cGAS and prevents its interaction with STING, thus inactivating the signaling pathway through the cGAS/STING/IRF3 axis. Consistently, addition of exogenous cGAMP to v65Rev-infected cells triggered the production of IFN-ß levels similar to those observed with v65Stop-infected cells, confirming that pp65 inactivation of IFN-ß production occurs at the cGAS level. Notably, within the first 24 h of HCMV infection, STING undergoes proteasome degradation independently of the presence or absence of pp65. Collectively, our data provide mechanistic insights into the interplay between HCMV pp65 and cGAS, leading to subsequent immune evasion by this prominent DNA virus.IMPORTANCE Primary human foreskin fibroblasts (HFFs) produce type I IFN (IFN-I) when infected with HCMV. However, we observed significantly higher IFN-ß levels when HFFs were infected with HCMV that was unable to express UL83-encoded pp65 (v65Stop), suggesting that pp65 (pUL83) may constitute a viral evasion factor. This study demonstrates that the HCMV tegument protein pp65 inhibits IFN-ß production by binding and inactivating cGAS early during infection. In addition, this inhibitory activity specifically targets cGAS, since it can be bypassed via the addition of exogenous cGAMP, even in the presence of pp65. Notably, STING proteasome-mediated degradation was observed in both the presence and absence of pp65. Collectively, our data underscore the important role of the tegument protein pp65 as a critical molecular hub in HCMV's evasion strategy against the innate immune response.

PYHIN genes as potential biomarkers for prognosis of human papillomavirus-positive or -negative head and neck squamous cell carcinomas.

The aim of the present study is to determine the expression levels of PYHIN (IFI16 and AIM2) and APOBEC3 (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) gene family members in a cohort of patients with head and neck squamous cell carcinoma (HNSCC) and assess their potential correlation with human papillomavirus (HPV) infection status, clinical characteristics, and survival. For this purpose, 34 HNSCC tissue specimens along with healthy surrounding mucosa were collected from patients surgically treated for HNSCC. Nucleic acids were isolated to assess the presence of HPV and the expression levels of selected molecular markers. Survival analysis was carried out using the Kaplan-Meier method. In HPV-negative (HPV-) HNSCCs, we detected low mRNA expression levels of IFI16, A3A, and A3B, whereas these genes were upregulated of 2-100 folds in HPV-positive (HPV+) tumors (p < 0.05). Interestingly, AIM2 gene expression levels were predominantly unchanged in HPV+ HNSCCs compared to their HPV- counterparts, in which AIM2 was predominantly upregulated (10% vs. 50% of patients). In HPV- tumors, upregulation of TP53, NOTCH1, PD-L1, and IFI16 correlated with lower occurrence of nodal metastases. On the other hand, the expression of APOBEC family members did not correlate with clinical characteristics. Regarding survival, patients with upregulated A3F gene expression had a worse prognosis, while patients without changes in A3H expression had a lower survival rate. In conclusion, our findings indicate that the innate immune sensors IFI16 and AIM2 and some APOBEC family members could be potentially used as biomarkers for disease outcome in HNSCC patients regardless of HPV presence.

Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability.

UNLABELLED: A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE: The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.