scholarly journals Identification of the Orthopoxvirus p4c Gene, Which Encodes a Structural Protein That Directs Intracellular Mature Virus Particles into A-Type Inclusions

2002 ◽  
Vol 76 (22) ◽  
pp. 11216-11225 ◽  
Author(s):  
Terry A. McKelvey ◽  
Stanley C. Andrews ◽  
Sara E. Miller ◽  
Caroline A. Ray ◽  
David J. Pickup
Keyword(s):  
Virus Replication ◽  
Cowpox Virus ◽  
Structural Protein ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Viral Morphogenesis ◽  
Vaccinia Virus Strain ◽  
Mature Virus

ABSTRACT The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.

scholarly journals A non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion

2020 ◽  
Author(s):  
Thomas Labadie ◽  
Polly Roy
Keyword(s):  
Extracellular Vesicles ◽  
Defense Mechanism ◽  
Degradation Process ◽  
Free Virus ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Super Infection

AbstractRecent developments on extracellular vesicles (EVs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. However, how non-enveloped viruses hijack cell machinery to promote non-lytic release in EVs, and their functional roles, remain to be clarified. Here we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and observed that the majority of viruses are released in EVs, both in vitro and in the blood of infected animals. Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic pH is neutralized upon the infection. Moreover, we report that secreted EVs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome.Author summaryRecent discoveries of non-enveloped virus secreted in EVs opened the door to new developments in our understanding of the transmission and pathogenicity of these viruses. In particular, how these viruses hijack the host cellular secretion machinery, and the role of these EVs compared with free-virus particles remained to be explored. Here, we tackled these two aspects, by studying BTV, an emerging arthropod-borne virus causing epidemics worldwide. We showed that this virus is mainly released in EVs, in vivo and in the blood of infected animals, and that inhibition of the cell degradation machinery decreases the release of infectious EVs, but not free-virus particles. We found that BTV must neutralize the pH of lysosomes, which are important organelles of the cell degradation machinery, for efficient virus release in EVs. Our results highlight unique features for a virus released in EVs, explaining how BTV transits in lysosomes without being degraded. Interestingly, we observed that EVs are more infectious than free-virus particles, but only free-viruses are able to overcome the super-infection exclusion, which is a common cellular defense mechanism. In conclusion, our study stresses the dual role played by both forms, free and vesicular, in the virus life cycle.

scholarly journals Cholesterol-modifying drugs in COVID-19

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Nathalie M Schmidt ◽  
Peter A C Wing ◽  
Jane A McKeating ◽  
Mala K Maini
Keyword(s):  
Immune Cell ◽  
Inflammatory Responses ◽  
The Elderly ◽  
Virus Infectivity ◽  
In Vivo Models ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Poor Outcomes

Abstract Infection with severe acute respiratory syndrom coronavirus 2 (SARS-CoV-2) is more likely to lead to poor outcomes in the elderly and those with cardiovascular disease, obesity or metabolic syndrome. Here, we consider mechanisms by which dyslipidaemia and the use of cholesterol-modifying drugs could influence the virus–host relationship. Cholesterol is essential for the assembly, replication and infectivity of enveloped virus particles; we highlight several cholesterol-modifying drugs with the potential to alter the SARS-CoV-2 life cycle that could be tested in in vitro and in vivo models. Although cholesterol is an essential component of immune cell membranes, excess levels can dysregulate protective immunity and promote exaggerated pulmonary and systemic inflammatory responses. Statins block the production of multiple sterols, oxysterols and isoprenoids, resulting in a pleiotropic range of context-dependent effects on virus infectivity, immunity and inflammation. We highlight antiviral, immunomodulatory and anti-inflammatory effects of cholesterol-modifying drugs that merit further consideration in the management of SARS-CoV-2 infection.

scholarly journals Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1294
Author(s):  
Robert Koban ◽  
Markus Neumann ◽  
Philipp P. Nelson ◽  
Heinz Ellerbrok
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Virus Replication ◽  
3D Culture ◽  
Human Keratinocytes ◽  
Infection Model ◽  
Cowpox Virus ◽  
Primary Human Keratinocytes

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.

scholarly journals Cowpox Virus Expresses a Novel Ankyrin Repeat NF-κB Inhibitor That Controls Inflammatory Cell Influx into Virus-Infected Tissues and Is Critical for Virus Pathogenesis

2009 ◽  
Vol 83 (18) ◽  
pp. 9223-9236 ◽  
Author(s):  
Mohamed Ragaa Mohamed ◽  
Masmudur M. Rahman ◽  
Amanda Rice ◽  
Richard W. Moyer ◽  
Steven J. Werden ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Virus Replication ◽  
Gene Knockout ◽  
Cowpox Virus ◽  
Iκb Kinase ◽  
Subsequent Degradation ◽  
Cellular Inhibitors ◽  
In Vivo Characterization

ABSTRACT Many pathogenic orthopoxviruses like variola virus, monkeypox virus, and cowpox virus (CPXV), but not vaccinia virus, encode a unique family of ankyrin (ANK) repeat-containing proteins that interact directly with NF-κB1/p105 and inhibit the NF-κB signaling pathway. Here, we present the in vitro and in vivo characterization of the targeted gene knockout of this novel NF-κB inhibitor in CPXV. Our results demonstrate that the vCpx-006KO uniquely induces a variety of NF-κB-controlled proinflammatory cytokines from infected myeloid cells, accompanied by a rapid phosphorylation of the IκB kinase complex and subsequent degradation of the NF-κB cellular inhibitors IκBα and NF-κB1/p105. Moreover, the vCpx-006KO virus was attenuated for virulence in mice and induced a significantly elevated cellular inflammatory process at tissue sites of virus replication in the lung. These results indicate that members of this ANK repeat family are utilized specifically by pathogenic orthopoxviruses to repress the NF-κB signaling pathway at tissue sites of virus replication in situ.

scholarly journals Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

2009 ◽  
Vol 83 (17) ◽  
pp. 8655-8661 ◽  
Author(s):  
Michael L. Grantham ◽  
Wai-Hong Wu ◽  
Erin N. Lalime ◽  
Maria E. Lorenzo ◽  
Sabra L. Klein ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Cysteine Residue ◽  
M2 Protein ◽  
Recombinant Viruses ◽  
Virus Particles

ABSTRACT The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

scholarly journals The formation and function of extracellular enveloped vaccinia virus

2002 ◽  
Vol 83 (12) ◽  
pp. 2915-2931 ◽  
Author(s):  
Geoffrey L. Smith ◽  
Alain Vanderplasschen ◽  
Mansun Law
Keyword(s):  
Vaccinia Virus ◽  
Cell Biology ◽  
Neutralizing Antibody ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Virus Life Cycle ◽  
And Function ◽  
Mature Virus ◽  
Cell Spread

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.

scholarly journals In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection

2012 ◽  
Vol 93 (6) ◽  
pp. 1269-1277 ◽  
Author(s):  
Kai Sen Tan ◽  
Farzad Olfat ◽  
Meng Chee Phoon ◽  
Jung Pu Hsu ◽  
Josephine L. C. Howe ◽  
...  
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
H1n1 Virus ◽  
Lung Damage ◽  
Structural Protein ◽  
Significant Difference ◽  
Antiviral Activities ◽  
Influenza H1n1

Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 103 and 104 p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.

scholarly journals Towards plant resistance to viruses using protein-only RNase P

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anthony Gobert ◽  
Yifat Quan ◽  
Mathilde Arrivé ◽  
Florent Waltz ◽  
Nathalie Da Silva ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Virus Replication ◽  
Yield Loss ◽  
Rnase P ◽  
Plant Viruses ◽  
Yellow Mosaic Virus ◽  
Resistance To Viruses ◽  
Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

scholarly journals Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

2001 ◽  
Vol 183 (4) ◽  
pp. 1423-1433 ◽  
Author(s):  
Susan R. Heimer ◽  
Harry L. T. Mobley
Keyword(s):  
Proteus Mirabilis ◽  
Accessory Proteins ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Nickel Ions ◽  
Catalytically Active ◽  
Tract Infections ◽  
Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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