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Human cytomegalovirus (HCMV) is a herpes virus with a long replication cycle. HCMV encoded long non-coding RNA termed RNA2.7 is the dominant transcript with a length of about 2.5kb, accounting for 25% of total viral transcripts. Studies have shown that HCMV RNA2.7 inhibits apoptosis caused by infection. The effect of RNA2.7 on other forms of cell death is still unclear. In this work, we found that RNA2.7 deletion significantly decreased the viability of HCMV-infected cells, while treatment with ferroptosis inhibitor Fer-1 rescued the infection-induced cell death, demonstrating an anti-ferroptosis role of RNA2.7. The results further showed that RNA2.7 inhibited ferroptosis via enhancing Ferritin Heavy Chain 1 (FTH1) and Solute Carrier Family 7 Member 11 (SLC7A11) expression in Erastin treated cells without involving other viral components. Pooled Genome-wide CRISPR screening revealed zinc finger protein 395 (ZNF395) as a new regulator repressing the expression of FTH1 and SLC7A11. HCMV RNA2.7 promoted proteasome-mediated degradation of ZNF395 that resulted in upregulation of FTH1 and SLC7A11 to inhibit ferroptosis, therefore maintain survival in host cells and complete replication of virus. Author summary: Human cytomegalovirus (HCMV) exhibits a latency that persists throughout life following the primary infection. Approximately 0.6% of newborns are HCMV-congenitally infected, 20–25% of which are symptomatic at birth or will develop long-term sequelae with a substantial public health impact. HCMV is slow-replicating virus that maintains the survival of the host cell to ensure virus replication. Several HCMV encoded proteins have been found to inhibit apoptosis and necroptosis following viral infection. However, the role of HCMV in other types of programmed cell death such as ferroptosis has not been clarified. In this study, we found that HCMV RNA2.7 inhibited ferroptosis independent on other viral components. Then we tried to explore how RNA2.7 inhibit ferroptosis and found RNA2.7 effectively increased ferritin and GSH contents to dampen ferroptosis. However, the regulation of RNA2.7 on ferritin and GSH contents was not depend on any previously reported regulators. Therefore, a pooled genome-wide CRISPR was employed and ZNF395 was determined as a newly regulator that promoted ferroptosis. Then, we confirmed that HCMV RNA2.7 promoted proteasome-mediated degradation of ZNF395 that resulted in upregulation of FTH1 and SLC7A11 to inhibit ferroptosis, therefore maintain survival in host cells and complete replication of virus. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV), a herpesvirus family member, is a large, complex enveloped virus. The activation of liver X receptor (LXR) can significantly inhibit the replication of HCMV and weaken the virulence of progeny virus (unpublished data). Our results showed activated LXR affected some important viral protein expression and reduced cholesterol content in HCMV infected cells and virus particles. To further clarify the influence of activated LXR on HCMV replication, HCMV assembly and maturation processes were studied by transmission electron microscopy (TEM) in HCMV infected foreskin fibroblasts treated with LXR agonist GW3965. Results showed that activated LXR could reduce the envelope integrity of maturating virions. The functional stage of activated LXR on viral envelope integrity was mainly at virus assembly compartment (VAC) mediated envelopment but not structurally complete virus nucleocapsid formation and the egress of nucleocapsid from the nucleus to the cytoplasm mediated by nuclear egress complex. Reduced cholesterol synthesis and viral protein expression might interfere with the VAC-mediated envelopment. The nucleocapsid and tegument proteins enter the VAC area for the secondary envelope, which was interfered with and resulted in the defective particle, thereby affecting the amount and infectivity of the mature virus. The results indicate that inhibition of HCMV maturation is one mechanism of activated LXR inhibiting virus replication in infected cells. [ABSTRACT FROM AUTHOR]

During infection with human cytomegalovirus (HCMV), overexpression of hcmv-miR-US33 can inhibit the lytic viral replication and down-regulate US29 mRNA. However, it remains unknown whether inhibition of viral replication by miR-US33 is mediated by down-regulation of expression of US29 or another host gene. Here, we identified the host gene Syntaxin3 ( STX3 ) to be a direct target of hcmv-miR-US33-5p using Hybrid-PCR and luciferase-reporter assays. It was further demonstrated that the levels of STX3 protein were down-regulated in hcmv-miR-US33-5p-overexpressing cells. Experiments with STX3-specific siRNA, or with an inhibitor of hcmv-miR-US33-5p confirmed that hcmv-miR-US33-5p-mediated inhibition of HCMV DNA synthesis and of viral replication are specifically mediated by down-regulation of STX3 expression. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play important roles in regulating various cellular processes in plants, animals, and viruses. This mechanism is also utilized by human cytomegalovirus (HCMV) in the process of infection and pathogenesis. The HCMV-encoded miRNA, hcmv-miR-US25-1-5p, was highly expressed during lytic and latent infections, and was found to inhibit viral replication. Identification of functional target genes of this microRNA is important in that it will enable a better understanding of the function of hcmv-miR-US25-1-5p during HCMV infection. In the present study, 35 putative cellular transcript targets of hcmv-miR-US25-1-5p were identified. Down-regulation of the targets YWHAE, UBB, NPM1, and HSP90AA1 by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. In addition, we showed that hcmv-miR-US25-1-5p could inhibit viral replication by interacting with these targets, the existence of which may impact virus replication directly or indirectly. [ABSTRACT FROM AUTHOR]