S-palmitoylation and sterol interactions mediate antiviral specificity of IFITM isoforms

Abstract Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITMs exhibit isoform-specific activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by molecular dynamic stimulations, nuclear magnetic resonance analysis in vitro and photoaffinity crosslinking in mammalian cells. These studies suggest that cholesterol can alter the conformation of IFITMs in membrane bilayers and directly interact with S-palmitoylated IFITMs in cells. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM isoform interactions with cholesterol in mammalian cells and specificity of antiviral activity towards IAV, SARS-CoV-2 and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction may influence host susceptibility to different viruses.

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A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune–epithelial interface

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2116853118 ◽

2021 ◽

Vol 119 (1) ◽

pp. e2116853118

Author(s):

Juliette Leon ◽

Daniel A. Michelson ◽

Judith Olejnik ◽

Kaitavjeet Chowdhary ◽

Hyung Suk Oh ◽

...

Keyword(s):

Influenza A ◽

Ebola Virus ◽

Multiorgan Failure ◽

Healthy Children ◽

Nonstructural Proteins ◽

Strong Response ◽

Mechanistic Insight ◽

Inflammatory Phenotype ◽

Transcriptional Induction

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2–specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

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Isolation and structures of the uprolides. I. Thirteen new cytotoxic cembranolides from the Caribbean gorgonian Euniceamammosa

Canadian Journal of Chemistry ◽

10.1139/v95-083 ◽

1995 ◽

Vol 73 (5) ◽

pp. 643-654 ◽

Cited By ~ 14

Author(s):

Abimael D. Rodríguez ◽

Ivette C. Piña ◽

Javier J. Soto ◽

Dalila R. Rojas ◽

Charles L. Barnes

Keyword(s):

Nuclear Magnetic Resonance Analysis ◽

X Ray Diffraction ◽

X Ray ◽

Correlation Studies ◽

Resonance Analysis ◽

Eupalmerin Acetate ◽

Chemical Correlation ◽

The Caribbean ◽

New Compounds

Thirteen new cembranolide diterpenoids, uprolides 3–15, have been isolated from the Caribbean gorgonian Euniceamammosa collected off the West coast of Puerto Rico. Several known metabolites, such as eupalmerin acetate (1) and eupalmerin (2), were also isolated from the same organism. The structures of the new compounds, which also showed modest in vitro cytotoxic activity, were assigned on the basis of extensive nuclear magnetic resonance analysis, chemical correlation studies, and by comparison with analogous spectral data from known cembranolide diterpenoids. One structure (3) was confirmed by X-ray diffraction analysis. Keywords: uprolides, Euniceamammosa, cytotoxicity, Caribbean, gorgonian.

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Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase

Journal of General Virology ◽

10.1099/vir.0.031492-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1650-1661 ◽

Cited By ~ 38

Author(s):

Ágnes Foeglein ◽

Eva M. Loucaides ◽

Manuela Mura ◽

Helen M. Wise ◽

Wendy S. Barclay ◽

...

Keyword(s):

Host Range ◽

Mammalian Cells ◽

Influenza A ◽

Transcriptional Activity ◽

Specific Activity ◽

Inhibitory Effects ◽

Influenza A Viruses ◽

Viral Polymerase ◽

Host Restriction ◽

Cellular Factors

Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as ‘signatures’ of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequence comparison followed by mutagenic analyses identified residues at known host-range-specific positions 271, 588 and 701 as well as a novel determinant at position 636 as contributors to host-specific activity of both PR8 and Turkey PB2 proteins. Furthermore, the correlation between poor transcriptional activity and slow diffusional mobility was maintained. However, activity did not obligatorily correlate with predicted surface charge of the 627 domain. Overall, our data support the hypothesis of a host nuclear factor that interacts with the viral polymerase and modulates its activity. While we cannot distinguish between positive and inhibitory effects, the data have implications for how such factors might operate.

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The Use of Antimalarial Drugs against Viral Infection

Microorganisms ◽

10.3390/microorganisms8010085 ◽

2020 ◽

Vol 8 (1) ◽

pp. 85 ◽

Cited By ~ 41

Author(s):

Sarah D’Alessandro ◽

Diletta Scaccabarozzi ◽

Lucia Signorini ◽

Federica Perego ◽

Denise P. Ilboudo ◽

...

Keyword(s):

Ebola Virus ◽

Viral Infections ◽

Antimalarial Drugs ◽

Drug Resistant ◽

Emerging Viruses ◽

Antimicrobial Drugs ◽

Artemisinin Derivatives ◽

Antiviral Activities

In recent decades, drugs used to treat malaria infection have been shown to be beneficial for many other diseases, including viral infections. In particular, they have received special attention due to the lack of effective antiviral drugs against new emerging viruses (i.e., HIV, dengue virus, chikungunya virus, Ebola virus, etc.) or against classic infections due to drug-resistant viral strains (i.e., human cytomegalovirus). Here, we reviewed the in vitro/in vivo and clinical studies conducted to evaluate the antiviral activities of four classes of antimalarial drugs: Artemisinin derivatives, aryl-aminoalcohols, aminoquinolines, and antimicrobial drugs.

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TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

Protein & Cell ◽

10.1007/s13238-020-00734-6 ◽

2020 ◽

Vol 11 (12) ◽

pp. 894-914

Author(s):

Nan Sun ◽

Li Jiang ◽

Miaomiao Ye ◽

Yihan Wang ◽

Guangwen Wang ◽

...

Keyword(s):

Influenza A ◽

Viral Infections ◽

Influenza Infection ◽

Type I ◽

Subsequent Formation ◽

Antiviral Immune Response ◽

Signaling Complex ◽

Deficient Mice

AbstractTripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.

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The biosynthesis of caerulomycins in Streptomyces caeruleus. Isolation of a new caerulomycin and incorporation of picolinic acid and glycerol into caerulomycin A

Canadian Journal of Chemistry ◽

10.1139/v88-031 ◽

1988 ◽

Vol 66 (1) ◽

pp. 191-194 ◽

Cited By ~ 17

Author(s):

Leo C. Vining ◽

A. Gavin McInnes ◽

Archibald W. McCulloch ◽

Donald G. Smith ◽

John A. Walter

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

High Efficiency ◽

Specific Activity ◽

Picolinic Acid ◽

Pyridyl Ring ◽

Nuclear Magnetic Resonance Analysis ◽

Glyceric Acid ◽

Carbon Precursor ◽

Resonance Analysis

Oxidation with permanganate of caerulomycin A labeled biosynthetically from [U-14C]lysine in cultures of Streptomyces caeruleus yielded picolinic acid with the same specific activity as the antibiotic. [3H]Picolinic acid was incorporated into caerulomycin A with high efficiency and the label was also found exclusively in picolinic acid after oxidation, implicating free picolinic acid as an intermediate in the pathway from lysine. The substituted pyridyl ring of caerulomycin was labeled by [2-13C, 1,3-14C]glycerol. 13C nuclear magnetic resonance analysis located the 13C predominantly at C-6, suggesting that C-5, C-6, and C-7 were derived from glycerol as an intact three-carbon precursor. Since no incorporation of [1-13C]glyceric acid was detected and [3-13C, 3-14C]methylglyoxal labeled the antibiotic only marginally, these compounds are probably not biosynthetic intermediates en route from glycerol. A new caerulomycin possessing an aldehyde in place of the aldoxime substituent at C-6 was isolated from culture broths and is postulated to be a late intermediate in the formation of caerulomycin A.

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Amino acids 473V and 598P of PB1 from an avian-origin influenza A virus contribute to polymerase activity, especially in mammalian cells

Journal of General Virology ◽

10.1099/vir.0.036434-0 ◽

2012 ◽

Vol 93 (3) ◽

pp. 531-540 ◽

Cited By ~ 41

Author(s):

Chen Xu ◽

Wei-Bin Hu ◽

Ke Xu ◽

Yun-Xia He ◽

Tong-Yan Wang ◽

...

Keyword(s):

Amino Acids ◽

Influenza A Virus ◽

Mammalian Cells ◽

Influenza A ◽

Polymerase Activity ◽

Amino Acid Residues ◽

Lower Growth ◽

Efficient Replication ◽

Avian Origin

It has been reported that the avian-origin influenza A virus PB1 protein (avian PB1) enhances influenza A virus polymerase activity in mammalian cells when it replaces the human-origin PB1 protein (human PB1). Characterization of the amino acid residues that contribute to this enhancement is needed. In this study, it was found that PB1 from an avian-origin influenza A virus [A/Cambodia/P0322095/2005, H5N1 (Cam)] could enhance the polymerase activity of an attenuated human isolated virus, A/WSN/33, carrying the PB2 K627E mutation (WSN627E) in vitro. Furthermore, 473V and 598P in the Cam PB1 were identified as the residues responsible for this enhanced activity. The results from recombinant virus experiments demonstrated the contribution of PB1 amino acids 473V and 598P to polymerase activity in mammalian cells and in mice. Interestingly, 473V is conserved in pH1N1 viruses from the 2009 pandemic. Substitution of 473V by leucine in pH1N1 PB1 led to a decreased viral polymerase activity and a lower growth rate in mammalian cells, suggesting that the PB1 473V also plays a role in maintaining efficient virus replication of the pH1N1 virus. Thus, it was concluded that two amino acids in avian-origin PB1, 473V and 598P, contribute to the polymerase activity of the H5N1 virus, especially in mammalian cells, and that 473V in PB1 also contributes to efficient replication of the pH1N1 strain.

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Association of Ebola Virus Matrix Protein VP40 with Microtubules

Journal of Virology ◽

10.1128/jvi.79.8.4709-4719.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4709-4719 ◽

Cited By ~ 54

Author(s):

Gordon Ruthel ◽

Gretchen L. Demmin ◽

George Kallstrom ◽

Melodi P. Javid ◽

Shirin S. Badie ◽

...

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Matrix Protein ◽

Ebola Virus ◽

Life Cycles ◽

Binding Motif ◽

Physical Interaction ◽

Tubulin Polymerization ◽

In Cells

ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

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Role of Ebola Virus Secreted Glycoproteins and Virus-Like Particles in Activation of Human Macrophages

Journal of Virology ◽

10.1128/jvi.79.4.2413-2419.2005 ◽

2005 ◽

Vol 79 (4) ◽

pp. 2413-2419 ◽

Cited By ~ 82

Author(s):

Victoria Wahl-Jensen ◽

Sabine K. Kurz ◽

Paul R. Hazelton ◽

Hans-Joachim Schnittler ◽

Ute Ströher ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Activation ◽

Ebola Virus ◽

Target Cells ◽

Primary Target ◽

Necrosis Factor Alpha ◽

Human Macrophages ◽

Virus Like Particles

ABSTRACT Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Δ-peptide), GP1, and GP1,2Δ. While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP1,2 in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.

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Synergistic Interferon Alpha-based Drug Combinations Inhibit SARS-CoV-2 and other viral Infections in Vitro

10.21203/rs.3.rs-189394/v1 ◽

2021 ◽

Author(s):

Aleksandr Ianevski ◽

Rouan Yao ◽

Eva Zusinaite ◽

Laura Lello ◽

Sainan Wang ◽

...

Keyword(s):

Interferon Alpha ◽

Influenza A ◽

Viral Infections ◽

Human Lung ◽

Therapeutic Potential ◽

A549 Cells ◽

Drug Dose ◽

Human Interferon ◽

Lung Epithelial

Abstract There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggered cell intrinsic and extrinsic antiviral responses and reduced replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human lung epithelial Calu-3 cells. However, IFNa alone was insufficient to completely abolish SARS-CoV-2 replication. Combinations of IFNa with camostat, remdesivir, EIDD-2801, cycloheximide or convalescent serum showed strong synergy and effectively inhibited SARS-CoV-2 infection. Additionally, we demonstrated synergistic antiviral activity of IFNa2a with pimodivir against influenza A virus (FluAV) infection in human lung epithelial A549 cells, as well as of IFNa2a with lamivudine against human immunodeficiency virus 1 (HIV-1) infection in human TZM-bl cells. Our results indicate that IFNa2a-based combinational therapies help to reduce drug dose and improve efficacy in comparison with monotherapies, making them attractive targets for further pre-clinical and clinical development.

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