ICP22/IE63 Mediated Transcriptional Regulation and Immune Evasion: Two Important Survival Strategies for Alphaherpesviruses

In the process of infecting the host, alphaherpesviruses have derived a series of adaptation and survival strategies, such as latent infection, autophagy and immune evasion, to survive in the host environment. Infected cell protein 22 (ICP22) or its homologue immediate early protein 63 (IE63) is a posttranslationally modified multifunctional viral regulatory protein encoded by all alphaherpesviruses. In addition to playing an important role in the efficient use of host cell RNA polymerase II, it also plays an important role in the defense process of the virus overcoming the host immune system. These two effects of ICP22/IE63 are important survival strategies for alphaherpesviruses. In this review, we summarize the complex mechanism by which the ICP22 protein regulates the transcription of alphaherpesviruses and their host genes and the mechanism by which ICP22/IE63 participates in immune escape. Reviewing these mechanisms will also help us understand the pathogenesis of alphaherpesvirus infections and provide new strategies to combat these viral infections.

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

Study On the Mechanism of LMP2A Maintaining Epstein-barr Virus Latency Infection Through Interaction With CXCR4

Epstein-Barr virus (EBV) belongs to the γ-herpesvirus subfamily and is the first human tumor virus to be discovered. The global adult infection rate exceeds 90%. EBV can participate in the regulation of multiple genes and multiple signal pathways through its latent genes. Many studies have reported that CXCR4 is involved in the development of gastric cancer, but there are few studies on the specific mechanism of its role in EBV-associated gastric cancer (EBVaGC). In this study, we explored the mechanism by which EBV-encoded products maintain EBV latent infection through interaction with CXCR4, and the role of CXCR4 in EBV positive cells. The results show that there is a positive feedback between the EBV-encoded products and CXCR4, and LMP2A can activate CXCR4 through the NF-κB pathway. In addition, CXCR4 can be fed back to LMP2A and EBNA1 through the ERK signaling pathway. At the same time, CXCR4 can promote the proliferation and migration of EBV-positive cells, reduce the expression of the immediate early protein BZLF1, and play an important role in maintaining the incubation period of EBV infection. These findings are conducive to the further targeted therapy of EBVaGC.

Data-Independent Acquisition-Based Proteome and Phosphoproteome Profiling Reveals Early Protein Phosphorylation and Dephosphorylation Events in Arabidopsis Seedlings upon Cold Exposure

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

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.

HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation

The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

CEUS-Based Radiomics Can Show Changes in Protein Levels in Liver Metastases After Incomplete Thermal Ablation

ObjectiveTo investigate the ability of contrast-enhanced ultrasound (CEUS)-based radiomics combined with machine learning to detect early protein changes after incomplete thermal ablation.MethodsHCT-26 colorectal adenoma cells were engrafted into the livers of 80 mice, which were randomly divided into 4 groups for palliative laser ablation. Changes in heat shock protein (HSP) and apoptosis-related protein expression in the tumors were assessed. SCID mice subjected to CEUS and ultrasonography were divided into training (n=56) and test (n=24) datasets. Then, 102 features from seven feature groups were extracted. We use the least absolute shrinkage and selection operator (LASSO) feature selection method to fit the machine learning classifiers. The feature selection methods and four classifiers were combined to determine the best prediction model.ResultsThe areas under the receiver-operating characteristic curves (AUCs) of the classifiers in the test dataset ranged from 0.450 to 0.932 (median: 0.721). The best score was obtained from the model in which the omics data of CEUS was analyzed in the arterial phase by random forest (RF) classification.ConclusionsA machine learning model, in which radiomics characteristics are extracted by multimodal ultrasonography, can accurately, rapidly and noninvasively identify protein changes after ablation.

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.