scholarly journals Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines

2015 ◽  
Vol 89 (14) ◽  
pp. 7373-7384 ◽  
Author(s):  
Benson Yee Hin Cheng ◽  
Emilio Ortiz-Riaño ◽  
Juan Carlos de la Torre ◽  
Luis Martínez-Sobrido
Keyword(s):  
Cultured Cells ◽  
Reporter Genes ◽  
Hemorrhagic Fever ◽  
Off Label Use ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccines ◽  
Expression Levels ◽  
Off Label

ABSTRACTSeveral members of theArenaviridaefamily cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuatedin vivoin a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome.IMPORTANCESeveral arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuatedin vivowhile conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.

scholarly journals Development of Live-Attenuated Arenavirus Vaccines Based on Codon Deoptimization

2015 ◽  
Vol 89 (7) ◽  
pp. 3523-3533 ◽  
Author(s):  
Benson Yee Hin Cheng ◽  
Emilio Ortiz-Riaño ◽  
Aitor Nogales ◽  
Juan Carlos de la Torre ◽  
Luis Martínez-Sobrido
Keyword(s):  
Mammalian Cells ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Hemorrhagic Fever ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccine ◽  
Codon Deoptimization

ABSTRACTArenaviruses have a significant impact on public health and pose a credible biodefense threat, but the development of safe and effective arenavirus vaccines has remained elusive, and currently, no Food and Drug Administration (FDA)-licensed arenavirus vaccines are available. Here, we explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse-genetics approaches to rescue a battery of recombinant LCMVs (rLCMVs) encoding CD NPs (rLCMV/NPCD) that showed attenuated growth kineticsin vitro. Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NPCD1and rLCMV/NPCD2were highly attenuatedin vivobut, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NPCD1and rLCMV/NPCD2were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy, and stability of a CD-based approach for developing live-attenuated vaccine candidates against human-pathogenic arenaviruses.IMPORTANCESeveral arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections, while antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and is associated with side effects. Here, we describe the generation of recombinant versions of the prototypic arenavirus LCMV encoding codon-deoptimized viral nucleoproteins (rLCMV/NPCD). We identified rLCMV/NPCD1and rLCMV/NPCD2to be highly attenuatedin vivobut able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated arenavirus vaccines.

scholarly journals The High Degree of Sequence Plasticity of the Arenavirus Noncoding Intergenic Region (IGR) Enables the Use of a Nonviral Universal Synthetic IGR To Attenuate Arenaviruses

2016 ◽  
Vol 90 (6) ◽  
pp. 3187-3197 ◽  
Author(s):  
Masaharu Iwasaki ◽  
Beatrice Cubitt ◽  
Brian M. Sullivan ◽  
Juan C. de la Torre
Keyword(s):  
Public Health ◽  
Intergenic Region ◽  
Protective Immunity ◽  
Hemorrhagic Fever ◽  
Health Problems ◽  
Wild Type ◽  
Lethal Challenge ◽  
Important Public Health ◽  
High Degree

ABSTRACTHemorrhagic fever arenaviruses (HFAs) pose important public health problems in regions where they are endemic. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the noncoding intergenic region (IGR) present in each arenavirus genome segment, the S and L segments (S-IGR and L-IGR, respectively), plays important roles in the control of virus protein expression and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFAs. In this study, we further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus lymphocytic choriomeningitis virus (rLCMVs), whose S-IGRs were replaced by the S-IGR of Lassa virus (LASV) or an entirely nonviral S-IGR-like sequence (Ssyn), are viable, indicating that the function of S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGRs were replaced by Ssyn or S-IGRs of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuatedin vivobut able to elicit protective immunity against a lethal challenge with wild-type LCMV. Our findings indicate that replacement of L-IGR by a nonviral Ssyn could serve as a universal molecular determinant of arenavirus attenuation.IMPORTANCEHemorrhagic fever arenaviruses (HFAs) cause high rates of morbidity and mortality and pose important public health problems in regions where they are endemic. Implementation of live-attenuated vaccines (LAVs) will represent a major step to combat HFAs. Here we document that the arenavirus noncoding intergenic region (IGR) has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCMVs containing nonviral synthetic IGRs. These rLCMVs were severely attenuatedin vivobut able to elicit protective immunity against a lethal challenge with wild-type LCMV. These nonviral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAVs to combat HFA.

scholarly journals Visualization of SARS-CoV-2 infection dynamic

2021 ◽  
Author(s):  
Chengjin Ye ◽  
Kevin Chiem ◽  
Jun-Gyu Park ◽  
Jesus Silvas ◽  
Desarey Morales Vasquez ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cultured Cells ◽  
Imaging System ◽  
Reporter Genes ◽  
Therapeutic Interventions ◽  
Wild Type Virus ◽  
Nucleocapsid Gene ◽  
Recombinant Viruses

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

scholarly journals N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
MR Fibi ◽  
P Hermentin ◽  
JU Pauly ◽  
L Lauffer ◽  
G Zettlmeissl
Keyword(s):  
Transient Expression ◽  
Cell Lines ◽  
Recombinant Human Erythropoietin ◽  
Biologically Active ◽  
Wild Type ◽  
Expression Levels ◽  
Human Erythropoietin ◽  
Culture Supernatants ◽  
Secretion Rates

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.

scholarly journals Groucho Oligomerization Is Required for Repression In Vivo

2004 ◽  
Vol 24 (10) ◽  
pp. 4341-4350 ◽  
Author(s):  
Haiyun Song ◽  
Peleg Hasson ◽  
Ze’ev Paroush ◽  
Albert J. Courey
Keyword(s):  
Target Genes ◽  
Cultured Cells ◽  
Coiled Coil ◽  
S2 Cells ◽  
Wild Type ◽  
Developmental Defects ◽  
Conserved Domain ◽  
Wing Disk ◽  
Single Motif

ABSTRACT Drosophila Groucho (Gro) is a member of a family of metazoan corepressors with widespread roles in development. Previous studies indicated that a conserved domain in Gro, termed the Q domain, was required for repression in cultured cells and mediated homotetramerization. Evidence presented here suggests that the Q domain contains two coiled-coil motifs required for oligomerization and repression in vivo. Mutagenesis of the putative hydrophobic faces of these motifs, but not of the hydrophilic faces, prevents the formation of both tetramers and higher order oligomers. Mutagenesis of the hydrophobic faces of both coiled-coil motifs in the context of a Gal4-Gro fusion protein prevents repression of a Gal4-responsive reporter in S2 cells, while mutagenesis of a single motif weakens repression. The finding that the repression directed by the single mutants depends on endogenous wild-type Gro further supports the idea that oligomerization plays a role in repression. Overexpression in the fly of forms of Gro able to oligomerize, but not of a form of Gro unable to oligomerize, results in developmental defects and ectopic repression of Gro target genes in the wing disk. Although the function of several corepressors is suspected to involve oligomerization, these studies represent one of the first direct links between corepressor oligomerization and repression in vivo.

scholarly journals Herpes Simplex Virus with Highly Reduced gD Levels Can Efficiently Enter and Spread between Human Keratinocytes

2001 ◽  
Vol 75 (21) ◽  
pp. 10309-10318 ◽  
Author(s):  
Mary T. Huber ◽  
Todd W. Wisner ◽  
Nagendra R. Hegde ◽  
Kimberley A. Goldsmith ◽  
Daniel A. Rauch ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cultured Cells ◽  
Human Keratinocytes ◽  
Hacat Cells ◽  
Wild Type ◽  
Simplex Virus ◽  
Cell Spread ◽  
Hsv 1

ABSTRACT The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

scholarly journals Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

2021 ◽  
Vol 17 (3) ◽  
pp. e1009356
Author(s):  
Takaaki Koma ◽  
Cheng Huang ◽  
Adrian Coscia ◽  
Steven Hallam ◽  
John T. Manning ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rational Design ◽  
Critical Role ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Wild Type ◽  
Virus Growth ◽  
Glycosylation Sites

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

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