Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (13) ◽  
Keyword(s):  
Host Cell ◽  
Immunofluorescence Microscopy ◽  
Viral Proteins ◽  
Kinase Activation ◽  
Gfp Fluorescence ◽  
Fluorescence Complementation

SARS-CoV-2 Biology Insights, Part I. Genome-wide structure and druggability: literature review (Preprint)

2020 ◽  
Author(s):  
Laura Lafon-Hughes
Keyword(s):  
Side Effects ◽  
Literature Review ◽  
Host Cell ◽  
Nucleoside Analogs ◽  
Viral Proteins ◽  
Therapeutic Strategies ◽  
Adverse Side Effects ◽  
Viral Proteases ◽  
Genome Wide ◽  
Therapeutic Alternatives

BACKGROUND COVID-19 pandemic prompts the study of coronavirus biology and search of putative therapeutic strategies. OBJECTIVE To compare SARS-CoV-2 genome-wide structure and proteins with other coronaviruses, focusing on putative coronavirus-specific or SARS-CoV-2 specific therapeutic designs. METHODS The genome-wide structure of SARS-CoV-2 was compared to that of SARS and other coronaviruses in order to gain insights, doing a literature review through Google searches. RESULTS There are promising therapeutic alternatives. Host cell targets could be modulated to hamper viral replication, but targeting viral proteins directly would be a better therapeutic design, since fewer adverse side effects would be expected. CONCLUSIONS Therapeutic strategies (Figure 1) could include the modulation of host targets (PARPs, kinases) , competition with G-quadruplexes or nucleoside analogs to hamper RDRP. The nicest anti-CoV options include inhibitors of the conserved essential viral proteases and drugs that interfere ribosome slippage at the -1 PRF site.

scholarly journals The association of viral proteins with host cell dynein components during virus infection

FEBS Journal ◽  
2011 ◽  
Vol 278 (17) ◽  
pp. 2997-3011 ◽  
Author(s):  
Javier Merino-Gracia ◽  
María F. García-Mayoral ◽  
Ignacio Rodríguez-Crespo
Keyword(s):  
Virus Infection ◽  
Host Cell ◽  
Viral Proteins

Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits

1993 ◽  
Vol 106 (2) ◽  
pp. 535-544 ◽  
Author(s):  
G. Maridor ◽  
P. Gallant ◽  
R. Golsteyn ◽  
E.A. Nigg
Keyword(s):  
Nuclear Localization ◽  
Immunofluorescence Microscopy ◽  
Cyclin A ◽  
S Phase ◽  
Catalytic Subunits ◽  
Kinase Activation ◽  
Indirect Immunofluorescence Microscopy ◽  
C Terminus ◽  
Dependent Protein ◽  
Cycle Regulation

Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.

scholarly journals Hijacking of Lipid Droplets by Hepatitis C, Dengue and Zika Viruses—From Viral Protein Moonlighting to Extracellular Release

2020 ◽  
Vol 21 (21) ◽  
pp. 7901 ◽  
Author(s):  
Alexandra P.M. Cloherty ◽  
Andrea D. Olmstead ◽  
Carla M.S. Ribeiro ◽  
François Jean
Keyword(s):  
Hepatitis C ◽  
Viral Infection ◽  
Host Cell ◽  
Lipid Droplets ◽  
Viral Proteins ◽  
Biological Properties ◽  
Protein Functions ◽  
Associated Proteins ◽  
Viral Lifecycle ◽  
Moonlighting Functions

Hijacking and manipulation of host cell biosynthetic pathways by human enveloped viruses are essential for the viral lifecycle. Flaviviridae members, including hepatitis C, dengue and Zika viruses, extensively manipulate host lipid metabolism, underlining the importance of lipid droplets (LDs) in viral infection. LDs are dynamic cytoplasmic organelles that can act as sequestration platforms for a unique subset of host and viral proteins. Transient recruitment and mobilization of proteins to LDs during viral infection impacts host-cell biological properties, LD functionality and canonical protein functions. Notably, recent studies identified LDs in the nucleus and also identified that LDs are transported extracellularly via an autophagy-mediated mechanism, indicating a novel role for autophagy in Flaviviridae infections. These developments underline an unsuspected diversity and localization of LDs and potential moonlighting functions of LD-associated proteins during infection. This review summarizes recent breakthroughs concerning the LD hijacking activities of hepatitis C, dengue and Zika viruses and potential roles of cytoplasmic, nuclear and extracellular LD-associated viral proteins during infection.

Racemization Hypothesis of COVID-19. Tip of the Iceberg

2020 ◽  
Keyword(s):  
Phase Transitions ◽  
Host Cell ◽  
Viral Infections ◽  
Proteolytic Enzymes ◽  
Viral Proteins ◽  
Cell Interaction ◽  
Cell Physiology ◽  
Viral Glycoprotein ◽  
Host Interaction ◽  
The Impact

The impact of viral infections on the central nervous system is widely known. Virus-related neuropsychiatric and neurobehavioral syndromes are caused by the distortion of cognitive, affective, behavioral, and perceptual domains. Although it is a commonly known phenomenon, the mechanism behind it is not well-understood. The contagious and deadly features of coronavirus disease 2019 (COVID-19) have been associated with the virus-host cell interaction at the molecular level. However, there is no reliable biomarker characterizing the disease progression. Studies of the structure, function, and evolution of coronavirus transmembrane spike glycoproteins (S-, N-, and E-proteins) suggest an essential role of protein chirality in virus-cell membrane interaction. The virus-host interaction is the subject of multidisciplinary research from the biochirality and systems biology, to cell physiology and non-equilibrium thermodynamics of phase transitions in proteins. At the protein level, virus-host interaction is modulated by the amino acid sequence of viral proteins and cellular metabolism. Enzymatic and spontaneous post-translational modifications (PTMs) are two mutually influential mechanisms governing the dynamics of virus and host cell proteome. Among them, phosphorylation and racemization are the most inter-related and studied. The spontaneous phase transitions within viral glycoprotein impacts the cell-entry capability of the virus. The spontaneous racemization is a particular and highly specific metabolic event in virus-cell interaction that is the focus of our attention. Many viral proteins are characterized by a high proportion of the serine (Ser) residues, which are the common target of the host-cell glycosylation, phosphorylation, and racemization, and proteolytic enzymes. Particularly, coronavirus N proteins were found to be phosphorylated at multiple Ser residues, a portion of which are shown to be phosphorylation-prone by the Ser-associated kinases. Since Ser is known as one of the most racemization prone amino acids, we promote an idea of the specific impact of spontaneous racemization at Ser residues on virus-host interaction.

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

1997 ◽  
Vol 43 (7) ◽  
pp. 625-632 ◽  
Author(s):  
M. S. Manocha ◽  
D. Xiong ◽  
V. Govindsamy
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Western Blot ◽  
Host Cell ◽  
Immunofluorescence Microscopy ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Primary Antibody ◽  
Rabbit Igg ◽  
Host Cell Surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

MSLVP: prediction of multiple subcellular localization of viral proteins using a support vector machine

2016 ◽  
Vol 12 (8) ◽  
pp. 2572-2586 ◽  
Author(s):  
Anamika Thakur ◽  
Akanksha Rajput ◽  
Manoj Kumar
Keyword(s):  
Support Vector Machine ◽  
Subcellular Localization ◽  
Host Cell ◽  
Subcellular Location ◽  
Viral Proteins ◽  
Support Vector

Knowledge of the subcellular location (SCL) of viral proteins in the host cell is important for understanding their function in depth.

scholarly journals Vaccinia Virus B1R Kinase Interacts with JIP1 and Modulates c-Jun-Dependent Signaling

2006 ◽  
Vol 80 (15) ◽  
pp. 7667-7675 ◽  
Author(s):  
Claudio R. Santos ◽  
Sandra Blanco ◽  
Ana Sevilla ◽  
Pedro A. Lazo
Keyword(s):  
Vaccinia Virus ◽  
Host Cell ◽  
Kinase Activity ◽  
Viral Proteins ◽  
Cell Interaction ◽  
Scaffold Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Potential Contribution ◽  
Mitogen Activated Protein ◽  
Host Cell Interaction

ABSTRACT Viruses have to adjust to the host cell to guarantee their life cycle and survival. This aspect of the virus-host cell interaction is probably performed by viral proteins, such as serine-threonine kinases, that are present early during infection. Vaccinia virus has an early Ser-Thr kinase, B1R, which, although required for successful viral infection, is poorly characterized regarding its effects on cellular proteins, and thus, its potential contribution to pathogenesis is not known. Signaling by mitogen-activated protein kinase (MAPK) is mediated by the assembly of complexes between these kinases and the JIP scaffold proteins. To understand how vaccinia virus B1R can affect the host, its roles in the cellular signaling by MAPK complexes and c-Jun activation have been studied. Independently of its kinase activity, B1R can interact with the central region of the JIP1 scaffold protein. The B1R-JIP1 complex increases the amount of MAPK bound to JIP1; thus, MKK7 and TAK1 either bind with higher affinity or bind more stably to JIP1, while there is an increase in the phosphorylation state of JNK bound to JIP1. The functional consequence of these more stable interactions is an increase in the activity of transcription factors, such as c-Jun, that respond to these complexes. Furthermore, B1R is also able to directly phosphorylate c-Jun in residues different from those targeted by JNK and, thus, B1R can also cooperate by an independent route in c-Jun activation. Vaccinia virus B1R can thus modulate the signaling of pathways that respond to cellular stress.

scholarly journals Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 2193-2200 ◽  
Author(s):  
Manjusha M. Kulkarni ◽  
Cheryl L. Olson ◽  
David M. Engman ◽  
Bradford S. McGwire
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Host Cell ◽  
Posttranslational Modification ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antiserum ◽  
Cell Infection ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes ◽  
Triton X

ABSTRACT The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

scholarly journals KSHV lytic mRNA is efficiently translated in the absence of eIF4F

2018 ◽  
Author(s):  
Eric S. Pringle ◽  
Carolyn-Ann Robinson ◽  
Nicolas Crapoulet ◽  
Andrea L-A. Monjo ◽  
Katrina Bouzanis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Host Cell ◽  
Translation Initiation ◽  
Viral Protein ◽  
Viral Proteins ◽  
Lytic Replication ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas

ABSTRACTHerpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5 ′ -m7GTP caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate protein synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation mechanisms support viral protein synthesis. Here, using polysome profiling analysis, we confirmed that eIF4F disassembly did not affect the efficient translation of viral mRNAs during lytic replication, whereas a large fraction of host mRNAs remained eIF4F-dependent. Lytic replication altered multiple host translation initiation factors (TIFs), causing caspase-dependent cleavage of eIF2α and eIF4G1 and decreasing levels of eIF4G2 and eIF4G3. Non-eIF4F TIFs NCBP1, eIF4E2 and eIF4G2 associated with actively translating messenger ribonucleoprotein (mRNP) complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production, suggesting that the virus does not exclusively rely on one of these alternative TIFs for efficient viral protein synthesis. METTL3, an N6-methyladenosine (m6A) methyltransferase that modifies mRNAs and influences translational efficiency, was dispensable for early viral gene expression and genome replication but required for late gene expression and virion production. METTL3 was also subject to caspase-dependent degradation during lytic replication, suggesting that its positive effect on KSHV late gene expression may be indirect. Taken together, our findings reveal extensive remodelling of TIFs during lytic replication, which may help sustain efficient viral protein synthesis in the context of host shutoff.IMPORTANCEViruses use host cell protein synthesis machinery to create viral proteins. Herpesviruses have evolved a variety of ways to gain control over this host machinery to ensure priority synthesis of viral proteins and diminished synthesis of host proteins with antiviral properties. We have shown that a herpesvirus called KSHV disrupts normal cellular control of protein synthesis. A host cell protein complex called eIF4F starts translation of most cellular mRNAs, but we observed it is dispensable for efficient synthesis of viral proteins. Several proteins involved in alternative modes of translation initiation were likewise dispensable. However, an enzyme called METTL3 that modifies mRNAs is required for efficient synthesis of certain late KSHV proteins and productive infection. We observed caspase-dependent degradation of several host cell translation initiation proteins during infection, suggesting that the virus alters pools of available factors to favour efficient viral protein synthesis at the expense of host protein synthesis.

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