scholarly journals Human cytomegalovirus interactome analysis identifies degradation hubs, domain associations and viral protein functions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Luis V Nobre ◽  
Katie Nightingale ◽  
Benjamin J Ravenhill ◽  
Robin Antrobus ◽  
Lior Soday ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Protein Interactions ◽  
Adaptor Protein ◽  
Host Cells ◽  
Virus Protein ◽  
Protein Functions ◽  
Mechanism Of Degradation ◽  
Human Proteins ◽  
Infected Cells

Human cytomegalovirus (HCMV) extensively modulates host cells, downregulating >900 human proteins during viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a mass spectrometry-based interactome analysis of 169 tagged, stably-expressed canonical strain Merlin HCMV proteins, and two non-canonical HCMV proteins, in infected cells. This identified a network of >3400 virus-host and >150 virus-virus protein interactions, providing insights into functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to SH3 domains of NCK Adaptor Protein-1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection.

scholarly journals Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2157
Author(s):  
Norbert Odolczyk ◽  
Ewa Marzec ◽  
Maria Winiewska-Szajewska ◽  
Jarosław Poznański ◽  
Piotr Zielenkiewicz
Keyword(s):  
Drug Development ◽  
Protein Interactions ◽  
Rna Virus ◽  
Antiviral Drug ◽  
Host Protein ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Short Peptides ◽  
Virus Protein ◽  
Positive Strand Rna

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.

scholarly journals Povidone-Iodine Attenuates Viral Replication in Ocular Cells: Implications for Ocular Transmission of RNA Viruses

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 753
Author(s):  
Sneha Singh ◽  
Onkar B. Sawant ◽  
Shahzad I. Mian ◽  
Ashok Kumar
Keyword(s):  
Epithelial Cells ◽  
Viral Replication ◽  
Povidone Iodine ◽  
Rna Viruses ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Host Cells ◽  
Eye Banking ◽  
Antiviral Effects ◽  
Infected Cells

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1β) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

scholarly journals Direct infection of CD34+ progenitor cells by human cytomegalovirus: evidence for inhibition of hematopoiesis and viral replication

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1277-1283 ◽  
Author(s):  
M Movassagh ◽  
J Gozlan ◽  
B Senechal ◽  
C Baillou ◽  
JC Petit ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Laboratory Strain ◽  
Inhibitory Effect ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Differentiated Cells ◽  
Cd34 Antigen ◽  
Liquid Suspension ◽  
Infected Cells

We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.

scholarly journals BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 466 ◽  
Author(s):  
Francesco Martelli ◽  
Zongsong Wu ◽  
Serena Delbue ◽  
Fabian Weissbach ◽  
Maria Giulioli ◽  
...  
Keyword(s):  
Viral Replication ◽  
Point Mutations ◽  
T Antigen ◽  
Host Cells ◽  
Replication Capacity ◽  
Viral Gene ◽  
New Host ◽  
Infected Cells ◽  
Multiple Sclerosis Patients ◽  
Coding Control

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

2016 ◽  
Author(s):  
Saisai Chen ◽  
Thomas Shenk ◽  
Maciej T. Nogalski
Keyword(s):  
Viral Replication ◽  
Host Cell ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
Receptor Expression ◽  
Receptor Activity ◽  
P2y2 Receptor ◽  
Infected Cells

AbstractHuman cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. The current study reveals a role for purinergic signaling in HCMV infection. The levels of several components of the purinergic signaling system, including the P2Y2 receptor, were altered in HCMV-infected fibroblasts. P2Y2 receptor RNA and protein are strongly induced following infection. Pharmacological inhibition of receptor activity or knockdown of receptor expression markedly reduced the production of infectious HCMV progeny. When P2Y2 activity was inhibited, the accumulation of most viral RNAs tested and viral DNA was reduced. In addition, the level of cytosolic calcium within infected cells was reduced when P2Y2 signaling was blocked. The HCMV-coded UL37x1 protein was previously shown to induce calcium flux from the smooth endoplasmic reticulum to the cytosol, and the present study demonstrates that P2Y2 function is required for this mobilization. We conclude that P2Y2 supports the production of HCMV progeny, possibly at multiple points within the viral replication cycle that interface with signaling pathways induced by the purinergic receptor.ImportanceHCMV infection is ubiquitous and can cause life-threatening disease in immunocompromised patients, debilitating birth defects in newborns, and has been increasingly associated with a wide range of chronic conditions. Such broad clinical implications result from the modulation of multiple host cell processes. This study documents that cellular purinergic signaling is usurped in HCMV-infected cells and that the function of this signaling axis is critical for efficient HCMV infection. Therefore, we speculate that blocking P2Y2 receptor activity has the potential to become an attractive novel treatment option for HCMV infection.

scholarly journals Human cytomegalovirus directly modulates expression of chemokine CCL2 (MCP-1) during viral replication

2013 ◽  
Vol 94 (11) ◽  
pp. 2495-2503 ◽  
Author(s):  
Stuart T. Hamilton ◽  
Gillian M. Scott ◽  
Zin Naing ◽  
William D. Rawlinson
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Immune Modulation ◽  
Early Stage ◽  
Cell Nuclei ◽  
Virus Dissemination ◽  
Ccl2 Expression ◽  
Infected Cells ◽  
Inducing Factors ◽  
Stage Infection

Human cytomegalovirus (CMV) infects monocytes and other haematopoietic progenitor cells which then act as reservoirs for latency and virus dissemination. The chemokine CCL2 (monocyte chemotactic protein-1 or MCP-1) exhibits potent chemotactic activity for monocytes and is a likely target for CMV-induced immunomodulation. In this study, we demonstrate CMV modulates CCL2 expression in MRC-5 fibroblasts with multiplicity-dependent kinetics, where CCL2 is upregulated during early stage infection, followed by CCL2 inhibition at late stage infection. This CMV-induced CCL2 modulation was dependent upon virus replication, as UV-inactivated virus did not elicit any changes in CCL2 levels. Dual immunofluorescence staining showed CMV strains AD169, purified AD169, Merlin, FIX WT (FLAG-US28/WT) and pUS28-deficient FIX (FIX-ΔUS28) all induced upregulation of CCL2 primarily within infected cells. Focal upregulation of CCL2 within FIX-ΔUS28-infected cells demonstrated intracellular CCL2 accumulation was independent of CCL2 sequestration by the CMV-encoded chemokine receptor US28. Infection with purified virus confirmed CMV-induced CCL2 upregulation was not due to any CCL2-inducing factors contained within non-purified virus stocks. The CMV-induced CCL2 expression kinetics occurred concurrently with modulation of the CCL2 transcriptional activators NF-κB, interferon regulatory factor 3 and cytokine IFN-β, independent of virus strain, and with the establishment of viral replication compartments within infected cell nuclei. This is the first report to our knowledge to demonstrate CMV modulation of CCL2 expression within infected cells during viral replication. This immune modulation may facilitate virus dissemination, establishment of latency and pathogenesis of CMV-induced host disease.

scholarly journals Comprehensive analysis of the host-virus interactome of SARS-CoV-2

2021 ◽  
Author(s):  
Zhen Chen ◽  
Chao Wang ◽  
Xu Feng ◽  
Litong Nie ◽  
Mengfan Tang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Viral Proteins ◽  
Host Cells ◽  
Virus Protein ◽  
N Protein ◽  
Protein Protein Interaction ◽  
Human Coronaviruses

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

scholarly journals Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

2002 ◽  
Vol 76 (24) ◽  
pp. 12676-12682 ◽  
Author(s):  
Toru Kubota ◽  
Noriko Yokosawa ◽  
Shin-ichi Yokota ◽  
Nobuhiro Fujii
Keyword(s):  
Signal Transduction Pathway ◽  
Adaptor Protein ◽  
Mumps Virus ◽  
Virus Protein ◽  
V Protein ◽  
Long Form ◽  
Infected Cells ◽  
Intracellular Target ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

scholarly journals Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells

2007 ◽  
Vol 81 (18) ◽  
pp. 10123-10136 ◽  
Author(s):  
George Sourvinos ◽  
Nina Tavalai ◽  
Anja Berndt ◽  
Demetrios A. Spandidos ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Spatial Organization ◽  
Human Fibroblasts ◽  
Nuclear Bodies ◽  
Immediate Early ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Pml Nuclear Bodies ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise colocalizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.

SARS-CoV-2 proteins bind heme and hemoglobin

2021 ◽  
Author(s):  
Salvatore Giovanni De-Simone ◽  
Guilherme Curty Lechuga ◽  
Franklin Souza-Silva ◽  
Carolina de Queiroz Sacramento ◽  
Monique Ramos de Oliveira Trugilho ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protein Interactions ◽  
Protoporphyrin Ix ◽  
In Silico Analysis ◽  
Viral Proteins ◽  
Vero Cells ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Primary Concern

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins’ possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobin’s porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin. In vitro analyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1mM suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.

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