Infected Cell Protein Research Articles

Screening of Goose Parvovirus Vp1 and Interacting Proteins in Infected Host Cells and Correlation with Goose Parvovirus Replication

Screening of Goose Parvovirus Vp1 and Interacting Proteins in Infected Host Cells and Correlation with Goose Parvovirus Replication

Membrane proteomic analysis identifies the polarity protein PARD3 as a novel antiviral protein against PEDV infection

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. PEDV could remodel host membrane structures for their replication, assembly and escape from host cells. However, little is known about the host membrane proteins of PEDV infection. In this study, we analyzed differentially abundant proteins (DAPs) between PEDV infection group and control group and identified the polarity protein PARD3 as one of the most significantly DAPs. PARD3 is implicated in the formation of tight junctions at epithelial cell-cell contacts. Then, we found that PEDV infection promoted the degradation of PARD3 via the ubiquitin proteasome pathway. Moreover, knockdown of PARD3 promoted the proliferation of PEDV. Further study showed that the downregulation of PARD3 altered the normal morphology of the tight junction proteins and promoted apical and basolateral virus proliferation. Tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection and PEDV affected the barrier functions by causing degradation of PARD3. Taken together, this work is the first time to investigate the membrane protein profile of PEDV-infected cells using quantitative proteomics and suggests that PARD3 could be a potential novel antiviral protein against PEDV infection. SignificanceMembrane proteins are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are changed during in response to environmental stress. However, membrane proteins are difficult to study because of their hydrophobicity. Membrane proteomic methods using mass spectrometry analysis have been developed and applied for the characterization of the plasma membrane and subcellular organelles of various virus infected cells. Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen of importance to the swine industry, causing high mortality in neonatal piglets. Because PEDV infected Vero cells can lead to significant changes in cell membrane morphology and form syncytial lesions. Here, we isolated the membrane proteins of PEDV infected and control cells and applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially abundant proteins (DAPs) in PEDV-infected Vero cells and confirmed the DAPs by performing RT-qPCR and Western blot analysis. Among these differential proteins, we focused on a down-regulated protein PARD3 which is important for cell tight junction and cell polarity. Loss of PARD3 can destroy the tight junction of cells and promote the proliferation of PEDV in the apical and basolateral sides. These findings will provide valuable information to better understand the mechanisms underlying the host defense responses to PEDV infection.

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.

Stress Induced Transcription Factors Transactivate the Herpes Simplex Virus 1 Infected Cell Protein 27 (ICP27) Transcriptional Enhancer.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons, including sensory neurons within trigeminal ganglia. During latency, lytic cycle viral gene expression is silenced. However, stressful stimuli can trigger reactivation from latency. The viral tegument protein, VP-16, transactivates all immediate early (IE) promoters during productive infection. Conversely, cellular factors are expected to trigger viral gene expression during early stages of reactivation from latency and in non-neuronal cells that do not support high levels of productive infection. The glucocorticoid receptor (GR), synthetic corticosteroid dexamethasone, and certain stress-induced transcription factors cooperatively transactivate infected cell protein 0 (ICP0) and ICP4 promoters. Since ICP27 protein expression is required for productive infection, we hypothesized that the ICP27 promoter is transactivated by stress-induced transcription factors. New studies have demonstrated that ICP27 enhancer sequences were transactivated by GR and Krüppel-like factor 15 (KLF15). Mutation of a consensus Sp1 binding site within ICP27 enhancer sequences impaired transactivation by GR and KLF15. Chromatin immunoprecipitation studies have demonstrated that GR and KLF15 occupy ICP27 promoter sequences during productive infection. Cells transfected with an ICP27 enhancer fragment revealed the GR and KLF15 occupancy of ICP27 enhancer sequences required the intact Sp1 binding site. Notably, GR and KLF15 form a feed-forward transcription loop in response to stress, suggesting these cellular factors promote viral replication following stressful stimuli.

Characterization of a thiourea derivative that targets viral transactivators of cytomegalovirus and herpes simplex virus type 1

Although currently available antivirals against certain herpesviruses are effective, the development of resistance during long-term use has necessitated the search for seed compounds that work against novel target molecules. In this report, we identified a thiourea derivative compound, 147B3, that inhibits the infection of human cytomegalovirus (HCMV) in fibroblasts and herpes simplex virus type 1 (HSV-1) in Vero cells at a 50% effective concentration of 0.5 μM and 1.9 μM, respectively. Characterization of the compound provided the following clues regarding its mode of action. 1) Time-of-addition and block-release assays showed that 147B3 behaved similarly to ganciclovir. 2) 147B3 reduced the expression of early and late but not immediate-early gene products and the accumulation of viral genomic DNA in both HCMV-infected and HSV-1-infected cells. 3) 147B3 inhibited the HCMV IE2-dependent activation of viral early gene promoters. 4) Four HSV-1 clones resistant to 147B3 were isolated and next-generation sequencing analysis of their genome DNA revealed that all of them had a mutation(s) in the infected cell protein 4 (ICP4) gene, which encodes a viral transcriptional factor. 5) Although 147B3 did not reduce the amount of ICP4 in an immunoblotting analysis, it changed the localization of the ICP4 from the speckles in the nuclei to diffused dots in the cytoplasm. 6) 147B3 did not affect the localization of promyelocytic leukemia (PML) bodies. Our findings suggest that 147B3 targets viral transactivators, potentially through their interaction with factors required for the viral gene expression system.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

SARS-CoV-2 triggers DNA damage response in Vero E6 cells

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.

SARS-CoV-2 spike promotes inflammation and apoptosis through autophagy by ROS-suppressed PI3K/AKT/mTOR signaling

BackgroundSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation. MethodsSARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses. ResultsAngiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.

Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway.

The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.

Simple and rapid high-throughput assay to identify HSV-1 ICP0 transactivation inhibitors

Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6β (wt) and dlx3.1-6β (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter:lacz expression cassette. A ≥10-fold difference in β-galactosidase activity was observed in cells infected with KOS6β compared to dlx3.1-6β, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z′-factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.

A Pioneer Transcription Factor and Type I Nuclear Hormone Receptors Synergistically Activate the Bovine Herpesvirus 1 Infected Cell Protein 0 (ICP0) Early Promoter.

Following bovine herpesvirus 1 (BoHV-1) acute infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia are an important site for latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Expression of two key viral transcriptional regulatory proteins, BoHV-1 infected cell protein 0 (bICP0) and bICP4, are regulated by sequences within the immediate early promoter (IEtu1). A separate early promoter also drives bICP0 expression, presumably to ensure sufficient levels of this important transcriptional regulatory protein. Productive infection and bICP0 early promoter activity are cooperatively transactivated by Krüppel-like factor 4 (KLF4) and a type I nuclear hormone receptor (NHR), androgen receptor, glucocorticoid receptor, or progesterone receptor. The bICP0 early promoter contains three separate transcriptional enhancers that mediate cooperative transactivation. In contrast to the IEtu1 promoter, the bICP0 early promoter lacks consensus type I NHR binding sites. Consequently, we hypothesized that KLF4 and Sp1 binding sites are essential for type I NHR and KLF4 to transactivate the bICP0 promoter. Mutating KLF4 and Sp1 binding sites in each enhancer domain significantly reduced transactivation by KLF4 and a type I NHR. Chromatin immunoprecipitation (ChIP) studies demonstrated that occupancy of bICP0 early promoter sequences by KLF4 and type I NHR is significantly reduced when KLF4 and/or Sp1 binding sites are mutated. These studies suggest that cooperative transactivation of the bICP0 E promoter by type I NHRs and a stress-induced pioneer transcription factor (KLF4) promote viral replication and spread in neurons or nonneural cells in reproductive tissue. IMPORTANCE Understanding how stressful stimuli and changes in the cellular milieu mediate viral replication and gene expression in the natural host is important for developing therapeutic strategies that impair virus transmission and disease. For example, bovine herpesvirus 1 (BoHV-1) reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone, which mimics the effects of stress. Furthermore, BoHV-1 infection increases the incidence of abortion in pregnant cows, suggesting that sex hormones stimulate viral growth in certain tissues. Previous studies revealed that type I nuclear hormone receptors (NHRs) (androgen, glucocorticoid, or progesterone) and a pioneer transcription factor, Krüppel-like factor 4 (KLF4), cooperatively transactivate the BoHV-1 infected cell protein 0 (bICP0) early promoter. Transactivation was mediated by Sp1 and/or KLF4 consensus binding sites within the three transcriptional enhancers. These studies underscore the complexity by which BoHV-1 exploits type I NHR fluctuations to enhance viral gene expression, replication, and transmission in the natural host.

Molecular characterization and isolation of Infectious laryngotracheitis virus (ILTV) strains causing outbreaks in layer chicken farms of Kalubia Province, Egypt

Infectious Laryngotracheitis (ILT) is an acute highly contagious respiratory disease of chickens. It has significant economic importance due to mortalities and the drop in egg production. In this study, seventy samples from different layer farms were collected from the outbreaks that occurred in Kalubia province, Egypt, at the period from February 2018 till May 2019 to detect ILTV by molecular characterization through polymerase chain reaction assay (PCR) as well as isolation on the specific pathogen free- embryonated chicken eggs (SPF-ECEs) through chorioallantoic membrane (CAM) route. Post mortem examination of infected chickens revealed hemorrhagic tracheitis with fibrino-hemorrhagic casts and caseated materials. The PCR revealed amplification of a 688bp fragment of Infected Cell Protein 4 (ICP4) gene. Following that, seven samples were sequenced and phylogenetically analyzed. Sequence analysis of the ICP4 gene of these samples revealed complete identity with TCO (tissue culture origin) vaccines and the vaccine-related strains, which were previously isolated from Giza, Sharkia, Kafrelsheikh, and Fayoum provinces through the years 2007 to 2018. Inoculation of ILTV PCR positive samples on SPF-ECE appeared as yellowish-white pock lesions on the inoculated CAM from the first passage. From these results, we can say that ILTV circulating in Egypt is a vaccinal strain that regains its virulence from the back passage in birds and causes outbreaks all over the country.

Regulation of Krüppel-Like Factor 15 Expression by Herpes Simplex Virus Type 1 or Bovine Herpesvirus 1 Productive Infection.

Expression of Krüppel-like factor 15 (KLF15), a stress-induced transcription factor, is induced during bovine herpesvirus 1 (BoHV-1) reactivation from latency, and KLF15 stimulates BoHV-1 replication. Transient transfection studies revealed that KLF15 and glucocorticoid receptor (GR) cooperatively transactivate the BoHV-1-immediate-early transcription unit 1 (IEtu1), herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0), and ICP4 promoters. The IEtu1 promoter drives expression of bICP0 and bICP4, two key BoHV-1 transcriptional regulatory proteins. Based on these studies, we hypothesized infection is a stressful stimulus that increases KLF15 expression and enhances productive infection. New studies demonstrated that silencing KLF15 impaired HSV-1 productive infection, and KLF15 steady-state protein levels were increased at late stages of productive infection. KLF15 was primarily localized to the nucleus following infection of cultured cells with HSV-1, but not BoHV-1. When cells were transfected with a KLF15 promoter construct and then infected with HSV-1, promoter activity was significantly increased. The ICP0 gene, and to a lesser extent, bICP0 transactivated the KLF15 promoter in the absence of other viral proteins. In contrast, BoHV-1 or HSV-1 encoded VP16 had no effect on KLF15 promoter activity. Collectively, these studies revealed that HSV-1 and BoHV-1 productive infection increased KLF15 steady-state protein levels, which correlated with increased virus production.

SYNCRIP facilitates porcine parvovirus viral DNA replication through the alternative splicing of NS1 mRNA to promote NS2 mRNA formation

Porcine Parvovirus (PPV), a pathogen causing porcine reproductive disorders, encodes two capsid proteins (VP1 and VP2) and three nonstructural proteins (NS1, NS2 and SAT) in infected cells. The PPV NS2 mRNA is from NS1 mRNA after alternative splicing, yet the corresponding mechanism is unclear. In this study, we identified a PPV NS1 mRNA binding protein SYNCRIP, which belongs to the hnRNP family and has been identified to be involved in host pre-mRNA splicing by RNA-pulldown and mass spectrometry approaches. SYNCRIP was found to be significantly up-regulated by PPV infection in vivo and in vitro. We confirmed that it directly interacts with PPV NS1 mRNA and is co-localized at the cytoplasm in PPV-infected cells. Overexpression of SYNCRIP significantly reduced the NS1 mRNA and protein levels, whereas deletion of SYNCRIP significantly reduced NS2 mRNA and protein levels and the ratio of NS2 to NS1, and further impaired replication of the PPV. Furthermore, we found that SYNCRIP was able to bind the 3′-terminal site of NS1 mRNA to promote the cleavage of NS1 mRNA into NS2 mRNA. Taken together, the results presented here demonstrate that SYNCRIP is a critical molecule in the alternative splicing process of PPV mRNA, while revealing a novel function for this protein and providing a potential target of antiviral intervention for the control of porcine parvovirus disease.

Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Chemotherapy agents stimulate dendritic cells against human colon cancer cells through upregulation of the transporter associated with antigen processing

Single immunotherapy fails to demonstrate efficacy in patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Research on immune reactions before and after systemic agents for mCRC is warranted. Our study examined cell line models to compare the expression of immune surface markers on colon cancer cells before and after chemotherapy agents. We also elucidated mechanisms underlying the effects of chemotherapy agents on immune surface markers. We used real-world clinical samples with NanoString analysis and the Perkin-Elmer Opal multiplex system. We established that chemotherapy agents, particularly 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, stimulated the expression of stimulatory MHC class I alleles through stimulation the pathway of transporters associated with antigen processing 1 and 2 (TAP1 and TAP2) in cell line models. Application of infected cell protein 47 (ICP-47), a specific inhibitor of the TAP1/TAP2, significantly inhibited expression of TAP1/TAP2 and also inhibited the expression of the downstream MHC class I. In the functional assay, SN-38 significantly promoted the phagocytosis of colon cancer cells by monocyte-derived dendritic cells (MoDCs). We confirmed that the expression of major histocompatibility complex (MHC) class I, significantly increased after first-line chemotherapy and targeted therapy in the samples of real-world patients with de novo mCRC. Our study provides new insights for novel immunotherapy combinations.

Structural biology of coronavirus ion channels

Viral infection compromises specific organelles of the cell and readdresses its functional resources to satisfy the needs of the invading body. Around 70% of the coronavirus positive-sense single-stranded RNA encodes proteins involved in replication, and these viruses essentially take over the biosynthetic and transport mechanisms to ensure the efficient replication of their genome and trafficking of their virions. Some coronaviruses encode genes for ion-channel proteins - the envelope protein E (orf4a), orf3a and orf8 - which they successfully employ to take control of the endoplasmic reticulum-Golgi complex intermediate compartment or ERGIC. The E protein, which is one of the four structural proteins of SARS-CoV-2 and other coronaviruses, assembles its transmembrane protomers into homopentameric channels with mild cationic selectivity. Orf3a forms homodimers and homotetramers. Both carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Orf8 is a very short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers. This review addresses the contribution of biophysical and structural biology approaches that unravel different facets of coronavirus ion channels, their effects on the cellular machinery of infected cells and some structure-functional correlations with ion channels of higher organisms.

Infectivity of Human Adenovirus Type 55 to Human Intestinal Cells

To examine the infectivity of human adenovirus type 55 (HAdV-55) in human intestinal cells. Caco-2 cells were cultured in vitro, and infected with HAdV-3, 7, 14 and 55. The expression of viral proteins in infected cells was detected with immunofluorescence method. The intracellular and supernatant viral DNA levels were determined with fluorescent quantitative PCR at different points of time. The level of infectious virus particles in the supernatant of Caco-2 cells was determined with adenovirus sensitive HEp-2 infection assay. Immunofluorescence assay showed positive result for the expression of HAdV-55 virus protein in Caco-2 cells 48 h post infection. HAdV-3, 7, 14, and 55 showed sustained replication and proliferation in Caco-2 cells. The level of viral DNA in infected cells and the supernatant increased with the infection time, and the viral DNA level of HAdV-55 was significantly higher than those of HAdV-3, 7 and 14. The infectious virus particles of HAdV-55 in Caco-2 supernatant were more than those of HAdV-3, 7 and 14, showing statistically significant difference ( P<0.05). Caco-2 cells were infected with low doses of virus (1×TCID 50), and the cytopathic effect (CPE) of HAdV-55 infection wells was more significant than that of HAdV-3, 7 and 14 infection wells. This study found that human intestinal cells were susceptible to HAdV-55, and the infection level was higher than that of other common respiratory infections caused by adenovirus types 3, 7 and 14.

Observation of arenavirus nucleoprotein heptamer assembly.

Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single‐stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self‐assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full‐length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 ± 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.