IE1 Expression Research Articles

Brain-resident memory cells (bT RM) and virus reactivation

Abstract Background Neuroimmune responses terminate MCMV acute infection, however, a subset of neurons still harbor latent viral genomes. Factors regulating viral reactivation remain to be elucidated. Methods Using RNAscope, IE1 nucleic acid was detected within brains at D5 and D30 p.i. Using real-time RT-PCR, we assessed expression of IE1, E1, and gB transcripts. Using multi-color flow cytometry, we assessed depletion of bT RMfollowing icv injection of either α-CD8 Ab or α-CD103-sap. Using luciferase-expressing transgenic mice, we longitudinally assessed reactivation of cre-MCMV after T-cell depletion using live small animal imaging. Explant assay was employed to study the role of bT RMin reactivation. Using Nanostring, we identified changes following bT RM-depletion. Results Brain infection was demonstrated by X-gal staining as well as IE1 staining using RNAscope at 5 d p.i. After 30 d p.i., the virus established latency, as indicated by absence of transcripts (IE1, E1, and gB). After establishment, we injected either α-CD8 Ab or α-CD103-sap into the brain and showed 90–95% T-cell depletion. We infected mice with cre-MCMV, which expresses Cre recombinase. We observed enhanced imaging signals indicative of IE promoter activity in depleted animals. Surprisingly, we efficiently recovered virus from untreated mice, but not from those depleted of bT RM. Furthermore, on Nanostring analysis, we identified gpnmband hmox1, upregulated 11.48- and 6.7-fold following bT RMdepletion. Conclusion When we depleted bT RM, there was transient IE expression, sufficient to activate surveying microglia. This eventually resulted in a tissue-wide anti-viral state. These data provide new insights into the role of bT RMin controlling reactivation. This project was supported by award numbers NS-038836 from the National Institute of Neurological Disorders and Stroke

Human Cytomegalovirus IE1 Impairs Neuronal Migration by Downregulating Connexin 43.

Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.

Human Cytomegalovirus IE2 Disrupts Neural Progenitor Development and Induces Microcephaly in Transgenic Mouse

Human cytomegalovirus (HCMV) is a significant contributor to congenital birth defects. Limited by the lack of animal models, the pathogenesis of neurological damage in vivo caused by HCMV infection and the role of individual viral genes remain to be elucidated. Immediate early (IE2) protein may play a function in neurodevelopmental problems caused by HCMV infection. Here, this study intended to investigate IE2’s long-term effects on development of the brain in IE2-expressing transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) aimed to observe the phenotype of postnatal mice. The expression of IE2 in transgenic mice was confirmed by PCR and Western blot technology. We collected mouse brain tissue at 2, 4, 6, 8, and 10 days postpartum to analyze the developmental process of neural stem cells by immunofluorescence. We discovered that transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) can reliably produce IE2 in the brain at various postpartum phases. Furthermore, we also observed the symptoms of microcephaly in postnatal transgenic mice, and IE2 can damage the amount of neural stem cells, prevent them from proliferating and differentiating, and activate microglia and astrocytes, creating an unbalanced environment in the brain’s neurons. In conclusion, we demonstrate that long-term expression of HCMV-IE2 can cause microcephaly through molecular mechanisms affecting the differentiation and development of neural stem cells in vivo. This work establishes a theoretical and experimental foundation for elucidating the molecular mechanism of fetal microcephaly brought by HCMV infection in throughout the period of neural development of pregnancy.Graphical

The sORF-Encoded Peptides, ATP Synthase Subunits, Facilitate WSSV Duplication in Shrimp.

Short open reading frames (sORFs) are a newly identified family of genes, and the functions of most sORF genes and their encoded peptides (SEPs) are still unknown. In this study, two ATP synthase subunits were identified in kuruma shrimp (Marsupenaeus japonicus) as SEPs, namely MjATP5I and MjATP5L. They were widely distributed in all of the tested tissues of shrimp and upregulated in hemocytes and intestines in response to WSSV challenge. The injection of recombinant proteins (rMjATP5I and rMjATP5L) increased the expression of Ie1 and Vp28, while the knockdown of MjATP5I and MjATP5L decreased the expression of Ie1 and Vp28. All of the results suggest that MjATP5I and MjATP5L were beneficial for WSSV replication. Further exploration found that MjATP5I and MjATP5L RNAi significantly improved the shrimp survival rates, reduced ATP production, and upregulated the expression of antimicrobial peptide genes post viral challenge, and the two ATPase subunits and Relish negatively regulated each other. These results reveal that MjATP5I and MjATP5L facilitated WSSV duplication by regulating the production of ATP contents and the expression of antimicrobial peptide genes in shrimp.

UL34 Deletion Restricts Human Cytomegalovirus Capsid Formation and Maturation.

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.

Human Cytomegalovirus Immediate Early Protein 2 Protein Causes Cognitive Disorder by Damaging Synaptic Plasticity in Human Cytomegalovirus-UL122-Tg Mice.

Human cytomegalovirus (HCMV) infection is very common in the human population all around the world. Although the majority of HCMV infections are asymptomatic, they can cause neurologic deficits. Previous studies have shown that immediate early protein 2 (IE2, also known as UL122) of HCMV is related with the cognitive disorder mechanism. Due to species isolation, a HCMV-infected animal model could not be established which meant a study into the long-term effects of IE2 on neural development could not be carried out. By establishing HCMV-UL122-Tg mice (UL122 mice), we explored the cognitive behavior and complexity of neuron changes in this transgenic UL122 mice that could consistently express IE2 protein at different ages (confirmed in both 6- and 12-month-old UL122 mice). In the Morris water maze, cognitive impairment was more pronounced in 12-month-old UL122 mice than in 6-month-old ones. At the same time, a decrease of the density of dendritic spines and branches in the hippocampal neurons of 12-month-old mice was observed. Moreover, long-term potentiation was showed to be impaired in 12-month-old UL122 mice. The expressions of several synaptic plasticity-regulated molecules were reduced in 12-month-old UL122 mice, including scaffold proteins postsynaptic density protein 95 (PSD95) and microtubule-associated protein 2 (MAP2). Binding the expression of IE2 was increased in 12-month-old mice compared with 6-month-old mice, and results of statistical analysis suggested that the cognitive damage was not caused by natural animal aging, which might exclude the effect of natural aging on cognitive impairment. All these results suggested that IE2 acted as a pathogenic regulator in damaging synaptic plasticity by downregulating the expression of plasticity-related proteins (PRPs), and this damage increased with aging.

LvCPG2 facilitated WSSV infection by interaction with VP26 and VP28

Chondroitin sulfate proteoglycans (CSP), widely distributed in extracellular matrices, have several important functions in vertebrates. In certain viruses, CSP acts as a receptor to promote infection. However, chondroitin proteoglycans lack sulfate are poorly understood in invertebrates. In this study, chondroitin proteoglycan 2 of Litopenaeus vannamei (LvCPG2) was cloned. The open reading frame of LvCPG2 cDNA is 2133 bp, which encodes a protein of 710 amino acids. LvCPG2 contained eight Chitin-binding domain type 2 (ChtBD2). LvCPG2 had the highest expression in lymphoid and significantly increased after WSSV challenge. The relative expression of IE1 and VP28, as well as the viral copy numbers were decreased significantly in LvCPG2-silenced shrimp. The far-western blotting result showed that LvCPG2 interacted with VP26 and VP28. Molecular docking complexes showed that N-terminal of LvCPG2 interacted with C-terminal VP26, while C-terminal of LvCPG2 combined with N-terminal of VP28. Flow cytometry analysis indicated that LvCPG2 could facilitate WSSV adhesion and penetration of shrimp hemocytes. Collectively, these findings suggested that LvCPG2 was involved in WSSV infection by interaction with VP26 and VP28.

Litopenaeus vannamei peroxiredoxin 2-like is involved in WSSV infection by interaction with wsv089 and VP26

White spot syndrome virus (WSSV) is one of the most dangerous pathogen in shrimp aquaculture, which can cause extremely high mortality of shrimp. A full understanding of virus-host interactions is important to prevent viral infection. In the present study, wsv089-interacting molecule Litopenaeus vannamei peroxiredoxins2-like (LvPrx2-L) was selected by the yeast two-hybrid (Y2H) method. The interaction between wsv089 and LvPrx2-L was confirmed by far-western blotting assay. Interestingly, a further study indicated that LvPrx2-L interacted with VP26, and the molecular docking analysis supported the interaction between LvPrx2-L and VP26. Tissues distribution assay showed that LvPrx2-L was detected in all sampled tissues. The highest expression of LvPrx2-L was appeared in hemocytes. Following WSSV challenge, LvPrx2-L mRNA transcripts were significantly increased in the hemocytes and gill. In addition, the relative expression of IE1 and VP28 were remarkably up-regulated in the hepatopancreas and intestines of LvPrx2-L-knockdown shrimp. Moreover, the cumulative survival rate was significantly lower in the LvPrx2-L- silenced group compared with the control and blank groups. Furthermore, LvPrx2-L could regulate the expression of proPO, crustin, ALF3, and CAT at the mRNA level. These findings would further deepen our understanding of WSSV-host interaction and shrimp antiviral response. All these data might useful for assessing the function of LvPrx2-L in the immune response of crustacean.

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers.

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

Valspodar limits human cytomegalovirus infection and dissemination

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that establishes a life-long infection affecting up to 80% of the US population. HCMV periodically reactivates leading to enhanced morbidity and mortality in immunosuppressed patients causing a range of complications including organ transplant failure and cognitive disorders in neonates. Therapeutic options for HCMV are limited to a handful of antivirals that target late stages of the virus life cycle and efficacy is often challenged by the emergence of mutations that confer resistance. In addition, these antiviral therapies may have adverse reactions including neutropenia in newborns and an increase in adverse cardiac events in HSCT patients. These findings highlight the need to develop novel therapeutics that target different steps of the viral life cycle. To this end, we screened a small molecule library against ion transporters to identify new antivirals against the early steps of virus infection. We identified valspodar, a 2nd-generation ABC transporter inhibitor, that limits HCMV infection as demonstrated by the decrease in IE2 expression of virus infected cells. Cells treated with increasing concentrations of valspodar over a 9-day period show minimal cytotoxicity. Importantly, valspodar limits HCMV plaque numbers in comparison to DMSO controls demonstrating its ability to inhibit viral dissemination. Collectively, valspodar represents a potential new anti-HCMV therapeutic that limits virus infection by likely targeting a host factor. Further, the data suggest that specific ABC transporters may participate in the HCMV life-cycle.

Hsp90 function is required for stable transcription of the baculovirus transactivator ie-1 gene

A molecular chaperone heat shock protein 90 (Hsp90) is required for efficient infection by several viruses. Hsp90 has been recently implicated in baculovirus infection, but its exact role remains obscure. This study investigated the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The 17-AAG treatment significantly decreased the production of budded viruses and occlusion bodies in BmNPV-infected Bombyx mori cultured cells. Immunoblot and SDS-PAGE analyses showed that the expression of early and delayed early gene products, DBP and BRO, was delayed and dysregulated, and the very late gene product POLH was almost completely diminished. RT-qPCR experiments revealed that 17-AAG treatment did not affect initiation of the immediate early gene ie-1 expression, but the expression decreased by ∼50 % during the late stage of infection. 17-AAG treatment also decreased ie-1 promoter activity by ∼50 %. In addition, the expression of delayed early and late genes was dysregulated and inhibited, respectively. These results indicated that Hsp90 function is required for stable ie-1 transcription. Inhibiting Hsp90 function negatively affects ie-1 expression, resulting in dysregulation of delayed early genes and a severe decrease in late and very late gene expression.

The role of autophagy in human cytomegalovirus IE2 expression.

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

Shrimp SIRT1 activates of the WSSV IE1 promoter independently of the NF-κB binding site

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1-silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1, VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent.

Modulation of NPV gene expression pattern and retention of RNAi- based antiviral activity in inbred transgenic silkworm

Infections due to Bombyx mori nucleopolyhedro virus (BmNPV) in commercially important silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) causes up to 50% loss to silk production in India. Increased resistance to NPV through introduction of double stranded RNA transgenes against multiple NPV genes is reported in B. mori. However, stability of the RNAi - mediated antiviral activity has not been determined after several generations of inbred transgenic B. mori. Hence, we examined the NPV multiplication rate and induction of the disease symptoms in NPV- infected transgenic and non-transgenic silkworm after 35 generations. Significantly higher multiplication rate of NPV supported by higher copy number of gp-41 gene was observed in non- transgenic larvae as compared to transgenic larvae. Expression of NPV genes, ie-1, lef1 and p74 enhanced significantly in non-transgenic larva as compared to transgenic larva after NPV infection. Strong positive correlation was observed in the expression pattern of NPV genes in transgenic larvae which was absent in non-transgenic larvae. This indicated co-regulation of NPV gene expression in the presence of transgenes.

Mechanism analysis of Anti-BmNPV resistant strain NC99R

Bombyx mori is a lepidopteran insect with important economic value. Bombyx mori nucleopolyhedrovirus (BmNPV) causes huge economic loss to silkworm industry in China every year. The objective of this study is to determine the anti-BmNPV mechanism of Bombyx mori strain NC99R, and to provide a basis for understanding the molecular mechanism of the silkworm resistance strain. The normal control Dazao (DZ) strain and the NC99R resistant strain were fed with occlusion bodies (OB). The median lethal dose (LD50) analysis of the DZ and NC99R showed that the LD50 of DZ was 1.2×10⁵ OBs/larva, while NC99R was 1.8×10⁶ OBs/larva. The LD50 of the NC99R was about 15 times higher than the DZ. The mortality of DZ and NC99R were analyzed, which were fed with 1×10⁶ OBs/larva and injection with 1×10⁶ BVs/larva. The results showed that the death peak of DZ was concentrated in the 4th to 6th day. And the death peak of NC99R was concentrated in the 6th to 8th day, with a delay of 1-2 days compared with the control. The BmNPV DNA copy number showed that the BmNPV genome in DZ proliferated rapidly. The copy number of BmNPV DNA in NC99R were increased slowly after oral infection and body injection. HE staining showed that midgut tissue has no significant difference between DZ and NC99R in the early stage of oral infection. At 96 h p.i., the nucleus of DZ midgut became larger and shedding. The NC99R had enlarged nuclei, but the cells were still arranged neatly. Finally, the expression of virus genes in different periods were analyzed by RT-PCR. The results indicated that the immediate early gene ie-1 expression levels began to down-regulate after 24 h p.i.. The early, late, and extremely late genes were also down-regulated, and finally maintained at a lower expression level.

The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection.

As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus (HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew (Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts (TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.

Human Cytomegalovirus Immediate Early 1 Protein Causes Loss of SOX2 from Neural Progenitor Cells by Trapping Unphosphorylated STAT3 in the Nucleus.

The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV infection to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here, we show that HCMV infection of NPCs results in loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 downregulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.IMPORTANCE Human cytomegalovirus (HCMV) infections are a leading cause of brain damage, hearing loss, and other neurological disabilities in children. We report that the HCMV proteins known as IE1 and IE2 target expression of human SOX2, a central pluripotency-associated transcription factor that governs neural progenitor cell (NPC) fate and is required for normal brain development. Both during HCMV infection and when expressed alone, IE1 causes the loss of SOX2 from NPCs. IE1 mediates SOX2 depletion by targeting STAT3, a critical upstream regulator of SOX2 expression. Our findings reveal an unanticipated mechanism by which a common virus may cause damage to the developing nervous system and suggest novel targets for medical intervention.

The 5' Untranslated Region of the Major Immediate Early mRNA Is Necessary for Efficient Human Cytomegalovirus Replication.

The human cytomegalovirus (HCMV) immediate early 1 (IE1) and IE2 proteins are critical regulators of virus replication. Both proteins are needed to efficiently establish lytic infection, and nascent expression of IE1 and IE2 is critical for reactivation from latency. The regulation of IE1 and IE2 protein expression is thus a central event in the outcome of HCMV infection. Transcription of the primary transcript encoding both IE1 and IE2 is well studied, but relatively little is known about the posttranscriptional mechanisms that control IE1 and IE2 protein synthesis. The mRNA 5' untranslated region (5' UTR) plays an important role in regulating mRNA translation. Therefore, to better understand the control of IE1 and IE2 mRNA translation, we examined the role of the shared 5' UTR of the IE1 and IE2 mRNAs (MIE 5' UTR) in regulating translation. In a cell-free system, the MIE 5' UTR repressed translation, as predicted based on its length and sequence composition. However, in transfected cells we found that the MIE 5' UTR increased the expression of a reporter gene and enhanced its association with polysomes, demonstrating that the MIE 5' UTR has a positive role in translation control. We also found that the MIE 5' UTR was necessary for efficient IE1 and IE2 translation during infection. Replacing the MIE 5' UTR with an unstructured sequence of the same length decreased IE1 and IE2 protein expression despite similar levels of IE1 and IE2 mRNA and reduced the association of the IE1 and IE2 mRNAs with polysomes. The wild-type MIE 5'-UTR sequence was also necessary for efficient HCMV replication. Together these data identify the shared 5' UTR of the IE1 and IE2 mRNAs as an important regulator of HCMV lytic replication.IMPORTANCE The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining factors that regulate IE1 and IE2 expression is important for understanding the molecular events controlling the HCMV replicative cycle. Here we identify a positive role for the MIE 5' UTR in mediating the efficient translation of the IE1 and IE2 mRNAs. This result is an important advance for several reasons. To date, most studies of IE1 and IE2 regulation have focused on defining events that regulate IE1 and IE2 transcription. Our work reveals that in addition to the regulation of transcription, IE1 and IE2 are also regulated at the level of translation. Therefore, this study is important in that it identifies an additional layer of regulation controlling IE1 and IE2 expression and thus HCMV pathogenesis. These translational regulatory events could potentially be targeted by novel antiviral therapeutics that limit IE1 and IE2 mRNA translation and thus inhibit lytic replication or prevent HCMV reactivation.

Protein and DNA evidences of HCMV infection in primary breast cancer tissues and metastatic sentinel lymph nodes.

Breast cancer is the leading cause of death in women worldwide. There are evidences that human cytomegalovirus (HCMV) infection is associated with several malignant tumors. This study aims to investigate the infection of human cytomegalovirus (HCMV) in a large sample of breast cancer patients, and conduct a correlation analysis of clinical and pathological factors, to provide evidence for whether HCMV infection is associated with breast cancer development, progression and metastasis. A total of 438 tissue samples (including breast cancer tissue, paracancerous tissue and sentinel lymph node [SLN] tissue) obtained from 146 patients who were diagnosed with breast cancer and intraoperatively underwent unilateral axillary SLN biopsy at the Affiliated Hospital of Qingdao University from June 2013 to June 2014 were included into this study. These tissue samples were divided into two groups: SLN positive group and SLN negative group. The clinical information is collated and numbered. Normal breast tissues of 40 patients with cyclomastopathy were taken as controls. The expressions of HCMV immediate-early (IE) and late antigen (LA) proteins of breast cancer tissues, paracancerous tissues, SLN tissues and normal breast tissues were analyzed by immunohistochemistry, and HCMV infection of the samples was graded according to the percentage of the positive cells, and HCMV IE2 mRNA expression was detected by reverse transcription polymerase chain reaction. The clinical data were collated and statistically analyzed. HCMV IE and LA proteins were highly expressed in all breast cancer tissue samples. IE proteins were detected in 47.9% (70/146) of paracancerous tissue samples, and LA proteins were detected in 53.4% (78/146) of paracancerous tissue samples. IE and LA proteins were expressed in 92.6% of metastatic SLN samples (62/68) and in most of the tumor cells. Inflammatory cells in 60% (42/70) of non-metastatic samples were positive for HCMV. HCMV DNA was present in 100% of breast cancer tissue samples, 50% of paracancerous tissue samples, and 91% of metastatic SLN samples; but this was not present in HCMV negative and non-metastatic SLN samples. Differences in HCMV infection, and estrogen receptor-α, progesterone receptor, Elston classification, Ki67 percentage, Her-2 and Luminal type and other clinical indicators were not statistically significant. HCMV infection is common in breast cancer tissues, paracancerous tissues and SLN tissues. The severity of HCMV infection varied markedly with tissue type. HCMV infection might be associated with metastasis and invasion of breast cancer. The expression of HCMV IE2 was associated to breast cancer and lymph node metastasis. The expression level of estrogen receptor-α was related to HCMV infection.

Inhibition of human cytomegalovirus immediate early gene expression and growth by a novel RNase P ribozyme variant.

We have previously engineered new RNase P-based ribozyme variants with improved in vitro catalytic activity. In this study, we employed a novel engineered variant to target a shared mRNA region of human cytomegalovirus (HCMV) immediate early proteins 1 (IE1) and 2 (IE2), which are essential for the expression of viral early and late genes as well as viral growth. Ribozyme F-R228-IE represents a novel variant that possesses three unique base substitution point mutations at the catalytic domain of RNase P catalytic RNA. Compared to F-M1-IE that is the ribozyme derived from the wild type RNase P catalytic RNA sequence, the functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently. In cultured cells, expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98–99% and in HCMV production reduction by 50,000 folds. In contrast, expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds. Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression. Our results provide direct evidence that engineered RNase P ribozymes, such as F-R228-IE, can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection. Moreover, these results suggest that F-R228-IE, with novel and unique mutations at the catalytic domain to enhance ribozyme activity, can be a candidate for the construction of effective agents for anti-HCMV therapy.