Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
1.
World J Microbiol Biotechnol ; 38(9): 161, 2022 Jul 14.
Article in English | MEDLINE | ID: covidwho-1930506

ABSTRACT

A violacein-producing bacterium was isolated from a mud sample collected near a hot spring on Kümbet Plateau in Giresun Province and named the GK strain. According to the phylogenetic tree constructed using 16S rRNA gene sequence analysis, the GK strain was identified and named Janthinobacterium sp. GK. The crude violacein pigments were separated into three different bands on a TLC sheet. Then violacein and deoxyviolacein were purified by vacuum liquid column chromatography and identified by NMR spectroscopy. According to the inhibition studies, the HIV-1 RT inhibition rate of 1 mM violacein from the GK strain was 94.28% and the CoV-2 spike RBD:ACE2 inhibition rate of 2 mM violacein was 53%. In silico studies were conducted to investigate the possible interactions between violacein and deoxyviolacein and three reference molecules with the target proteins: angiotensin-converting enzyme 2 (ACE2), HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain. Ligand violacein binds strongly to the receptor ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -9.94 kcal/mol, -9.32 kcal/mol, and -8.27 kcal/mol, respectively. Deoxyviolacein strongly binds to the ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -10.38 kcal/mol, -9.50 kcal/mol, and -8.06 kcal/mol, respectively. According to these data, violacein and deoxyviolacein bind to all the receptors quite effectively. SARS-CoV-2 spike protein and HIV-1-RT inhibition studies with violacein and deoxyviolacein were performed for the first time in the literature.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , HIV-1 , Indoles , Spike Glycoprotein, Coronavirus , COVID-19/metabolism , COVID-19/virology , HIV-1/metabolism , Indoles/metabolism , Indoles/pharmacology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Protein Binding , RNA, Ribosomal, 16S , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
2.
J Biol Chem ; 298(5): 101924, 2022 05.
Article in English | MEDLINE | ID: covidwho-1778266

ABSTRACT

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA-protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA-protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA-protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA-protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.


Subject(s)
High-Throughput Nucleotide Sequencing , Proteins , RNA, Viral , HIV-1/genetics , HIV-1/metabolism , Host Microbial Interactions , Humans , Proteins/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics
3.
Viruses ; 13(10)2021 09 26.
Article in English | MEDLINE | ID: covidwho-1485180

ABSTRACT

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4+ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4+ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4+ T cells.


Subject(s)
HIV Infections/metabolism , Hyaluronan Receptors/metabolism , Leukosialin/metabolism , Membrane Glycoproteins/metabolism , Cell Membrane/metabolism , HIV Infections/genetics , HIV-1/genetics , HIV-1/metabolism , HIV-1/pathogenicity , Host-Pathogen Interactions , Humans , Hyaluronan Receptors/genetics , Leukosialin/genetics , Membrane Glycoproteins/genetics , Membrane Proteins/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Virion/metabolism , Virus Assembly , Virus Attachment , gag Gene Products, Human Immunodeficiency Virus/metabolism
4.
Viruses ; 13(2)2021 02 10.
Article in English | MEDLINE | ID: covidwho-1395005

ABSTRACT

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Subject(s)
Capsid/metabolism , Cell Nucleus/virology , HIV-1/physiology , Active Transport, Cell Nucleus , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Nucleus/metabolism , HIV-1/chemistry , HIV-1/metabolism , Models, Biological , Nuclear Pore Complex Proteins/metabolism , Reverse Transcription , Virus Integration , Virus Replication
5.
Int J Mol Sci ; 22(10)2021 May 17.
Article in English | MEDLINE | ID: covidwho-1383880

ABSTRACT

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , HIV-1/metabolism , Herpesviridae/metabolism , SARS-CoV-2/metabolism , Ebolavirus/metabolism , Endocytosis , Humans , Nipah Virus/metabolism , Protein Transport , Virus Release , Virus Replication
6.
Viruses ; 13(7)2021 06 30.
Article in English | MEDLINE | ID: covidwho-1287278

ABSTRACT

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.


Subject(s)
Host-Pathogen Interactions , Membrane Proteins/genetics , Viral Envelope Proteins , Virion/metabolism , Viruses/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Membrane Proteins/immunology , Retroviridae/classification , Retroviridae/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Virion/genetics , Virus Internalization , Viruses/chemistry , Viruses/classification , Viruses/genetics
7.
Int J Mol Sci ; 22(11)2021 May 26.
Article in English | MEDLINE | ID: covidwho-1256559

ABSTRACT

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.


Subject(s)
Ceramides/metabolism , HIV-1/metabolism , Influenza A virus/metabolism , SARS-CoV-2/metabolism , Virus Diseases/metabolism , Virus Diseases/virology , Apoptosis/drug effects , Apoptosis/immunology , Ceramides/chemistry , Gene Expression Regulation, Viral , HIV-1/pathogenicity , Humans , Immunomodulation , Influenza A virus/pathogenicity , SARS-CoV-2/pathogenicity , Virion/growth & development , Virus Diseases/immunology , Virus Internalization , Virus Replication/drug effects , Virus Replication/immunology
8.
Protein Expr Purif ; 181: 105837, 2021 05.
Article in English | MEDLINE | ID: covidwho-1057206

ABSTRACT

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.


Subject(s)
Glutamate-Ammonia Ligase/genetics , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Animals , CHO Cells , CRISPR-Cas Systems , Codon , Cricetulus , Gene Knockdown Techniques , HEK293 Cells , Humans , Protein Sorting Signals , Recombinant Proteins/metabolism
9.
J Virol ; 94(21)2020 10 14.
Article in English | MEDLINE | ID: covidwho-709870

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/physiology , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/metabolism , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , HEK293 Cells , HIV-1/genetics , HIV-1/metabolism , Humans , Leukemia Virus, Murine , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/metabolism , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Virion/genetics , Virion/immunology , Virion/metabolism , Virus Internalization
10.
Nat Commun ; 11(1): 2688, 2020 05 27.
Article in English | MEDLINE | ID: covidwho-432476

ABSTRACT

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.


Subject(s)
Glycoproteins/immunology , Middle East Respiratory Syndrome Coronavirus , Polysaccharides , Spike Glycoprotein, Coronavirus/immunology , Viral Fusion Proteins/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Glycoproteins/chemistry , Glycoproteins/ultrastructure , Glycosylation , HEK293 Cells , HIV-1/immunology , HIV-1/metabolism , Humans , Immune Evasion/physiology , Lassa virus/immunology , Lassa virus/metabolism , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Orthomyxoviridae/immunology , Orthomyxoviridae/metabolism , Polysaccharides/chemistry , Polysaccharides/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/ultrastructure , Viral Proteins/chemistry , Viral Proteins/immunology , Viral Proteins/ultrastructure
SELECTION OF CITATIONS
SEARCH DETAIL