scholarly journals MAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

2021 ◽  
Author(s):  
Ethan Laudermilch ◽  
Kartik Chandran
Keyword(s):  
Vaccinia Virus ◽  
Viral Genome ◽  
Point Mutations ◽  
Single Step ◽  
Viral Propagation ◽  
New Strategy ◽  
Plaque Purification ◽  
Infected Cells ◽  
Cancer Immunotherapies

AbstractVaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, a highly inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to rapidly introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.Graphical AbstractOverview of MAVERICC. Conceptual overview of the approach outlined in this manuscript. To make VACV recombinants, the parental virus is first purified and vDNA is isolated with phenol:chloroform extraction. This purified vDNA is then treated with Cas9 enzyme and sgRNAs that are directed to a specific locus in the VACV genome. The cleaved vDNA is then transfected into FWPV-infected BSC-40 cells along with a transfer amplicon containing an insertion or mutation of interest flanked by homologous sequences. Recombination is allowed to occur for 5-7 days, during which time the cleaved vDNA is healed by the transfer amplicon, thus editing the VACV genome, and packaged into infectious viral particles. Individual plaques are grown up and rVACVs are isolated after a single round of plaque purification. Image was created with Biorender.com.

scholarly journals Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

2009 ◽  
Vol 83 (13) ◽  
pp. 6464-6476 ◽  
Author(s):  
Yao-Cheng Ching ◽  
Che-Sheng Chung ◽  
Cheng-Yen Huang ◽  
Yu Hsia ◽  
Yin-Liang Tang ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Fusion ◽  
Disulfide Bond ◽  
Protein Complex ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
In Vitro Mutagenesis ◽  
Disulfide Bond Formation ◽  
Infected Cells

ABSTRACT Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

scholarly journals By Binding CD80 and CD86, the Vaccinia Virus M2 Protein Blocks Their Interactions with both CD28 and CTLA4 and Potentiates CD80 Binding to PD-L1

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Patricia Kleinpeter ◽  
Christelle Remy-Ziller ◽  
Eline Winter ◽  
Murielle Gantzer ◽  
Virginie Nourtier ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccinia Virus ◽  
Cytotoxic T Lymphocyte ◽  
Myxoma Virus ◽  
Parental Virus ◽  
M2 Protein ◽  
First Results ◽  
Infected Cells

ABSTRACTIn this article we report that the M2 protein encoded by the vaccinia virus is secreted as a homo-oligomer by infected cells and binds two central costimulation molecules, CD80 (B7-1) and CD86 (B7-2). These interactions block the ligation of the two B7 proteins to both soluble CD28 and soluble cytotoxic T-lymphocyte associated protein 4 (CTLA4) but favor the binding of soluble PD-L1 to soluble CD80. M2L gene orthologues are found in several other poxviruses, and the B7-CD28/CTLA4 blocking activity has been identified for several culture supernatants of orthopoxvirus-infected cells and for a recombinant myxoma virus M2 protein homolog (i.e., Gp120-like protein, or Gp120LP). Overall, these data indicate that the M2 poxvirus family of proteins may be involved in immunosuppressive activities broader than the NF-κB inhibition already reported (R. Gedey, X. L. Jin, O. Hinthong, and J. L. Shisler, J Virol 80:8676–8685, 2006, https://doi.org/10.1128/JVI.00935-06). A Copenhagen vaccinia virus with a deletion of the nonessential M2L locus was generated and compared with its parental virus. This M2L-deleted vaccinia virus, unlike the parental virus, does not generate interference with the B7-CD28/CTLA4/PD-L1 interactions. Moreover, this deletion did not affect any key features of the virus (in vitroreplication, oncolytic activitiesin vitroandin vivo,and intratumoral expression of a transgene in an immunocompetent murine model). Altogether, these first results suggest that the M2 protein has the potential to be used as a new immunosuppressive biotherapeutic and that the M2L-deleted vaccinia virus represents an attractive new oncolytic platform with an improved immunological profile.IMPORTANCEThe vaccinia virus harbors in its genome several genes dedicated to the inhibition of the host immune response. Among them, M2L was reported to inhibit the intracellular NF-κB pathway. We report here several new putative immunosuppressive activities of M2 protein. M2 protein is secreted and binds cornerstone costimulatory molecules (CD80/CD86). M2 binding to CD80/CD86 blocks their interaction with soluble CD28/CTLA4 but also favors the soluble PD-L1-CD80 association. These findings open the way for new investigations deciphering the immune system effects of soluble M2 protein. Moreover, a vaccinia virus with a deletion of its M2L has been generated and characterized as a new oncolytic platform. The replication and oncolytic activities of the M2L-deleted vaccinia virus are indistinguishable from those of the parental virus. More investigations are needed to characterize in detail the immune response triggered against both the tumor and the virus by this M2-defective vaccinia virus.

scholarly journals Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

2009 ◽  
Vol 54 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Olivier Delelis ◽  
Sylvain Thierry ◽  
Frédéric Subra ◽  
Françoise Simon ◽  
Isabelle Malet ◽  
...  
Keyword(s):  
Viral Genome ◽  
Effective Concentration ◽  
Coding Region ◽  
Delayed Emergence ◽  
Infected Cells ◽  
High Level ◽  
Emergence Of Resistance ◽  
Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

scholarly journals High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells

2003 ◽  
Vol 77 (13) ◽  
pp. 7281-7290 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Genetic Recombination ◽  
Virus Genome ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Plaque Purification ◽  
Infected Cells ◽  
Clone And Express ◽  
Recombination Reactions ◽  
Multiple Pcr

ABSTRACT Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 106 PFU/μg of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.

scholarly journals Characterization of the Vaccinia Virus A35R Protein and Its Role in Virulence

2006 ◽  
Vol 80 (1) ◽  
pp. 306-313 ◽  
Author(s):  
Rachel L. Roper
Keyword(s):  
Vaccinia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Growth ◽  
Protein Band ◽  
Mutant Virus ◽  
Translation System ◽  
Wild Type ◽  
Infected Cells

ABSTRACT The vaccinia virus A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. Western blotting with anti-A35R peptide antibodies indicated that the protein is expressed early in infection and resolved as a single sharp band of ∼23 kDa, slightly higher than the 20 kDa predicted from its sequence. The protein band appeared to be the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whether expressed in an in vitro transcription/translation system without microsomes or expressed in infected cells, suggesting that it was not glycosylated. A mutant virus with the A35R gene deleted (vA35Δ) formed wild-type-sized plaques on all cell lines tested (human, monkey, mouse, and rabbit); thus, A35R is not required for replication and does not appear to be a host range gene. Although the A35R protein is hydrophobic, it is unlikely to be an integral membrane protein, as it partitioned to the aqueous phase during TX-114 partitioning. The protein could not be detected in virus-infected cell supernatants. A35R localized intracellularly to the virus factories, where the first stages of morphogenesis occur. The vA35Δ mutant formed near-normal levels of the various morphogenic stages of infectious virus particles and supported normal acid-induced fusion of virus-infected cells. Despite normal growth and morphogenesis in vitro, the vA35Δ mutant virus was attenuated in intranasal challenge of mice compared to wild-type and A35R rescue virus. Thus, the intracellular A35R protein plays a role in virulence. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence mechanism.

scholarly journals A cytomegaloviral protein reveals a dual role for STAT2 in IFN-γ signaling and antiviral responses

2005 ◽  
Vol 201 (10) ◽  
pp. 1543-1553 ◽  
Author(s):  
Albert Zimmermann ◽  
Mirko Trilling ◽  
Markus Wagner ◽  
Manuel Wilborn ◽  
Ivan Bubic ◽  
...  
Keyword(s):  
Biological Significance ◽  
Receptor Expression ◽  
Type I ◽  
Antiviral Responses ◽  
Type I Ifns ◽  
New Strategy ◽  
Infected Cells ◽  
Ifn Γ

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (α/β), but it had a much more dramatic effect on type II IFNs (γ) in vitro and in vivo. A comparative analysis of M27+ and M27− MCMV revealed that the antiviral efficiency of IFN-γ was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-γ. This effect required IFN receptor expression and was independent of type I IFNs. IFN-γ induced increasing levels of tyrosine-phosphorylated STAT2 in M27− MCMV-infected cells that were essential for the antiviral potency of IFN-γ. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-γ responses.

scholarly journals Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

2015 ◽  
Vol 112 (29) ◽  
pp. 9010-9015 ◽  
Author(s):  
Kendra E. Leigh ◽  
Mayuri Sharma ◽  
My Sam Mansueto ◽  
Andras Boeszoermenyi ◽  
David J. Filman ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Structural Information ◽  
Point Mutations ◽  
Nuclear Lamina ◽  
Protein Protein Interaction ◽  
Nuclear Egress ◽  
Peptide Binding Site ◽  
Infected Cells ◽  
Nmr Methods

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1–168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50–UL53 binding in vitro, eliminated UL50–UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein–protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

scholarly journals Novel Modified Vaccinia Virus Ankara Vector Expressing Anti-apoptotic GeneB13RDelays Apoptosis and Enhances Humoral Responses

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Lynette S. Chea ◽  
Linda S. Wyatt ◽  
Sailaja Gangadhara ◽  
Bernard Moss ◽  
Rama R. Amara
Keyword(s):  
Vaccinia Virus ◽  
Vaccine Development ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Antibody Titers ◽  
Infected Cells ◽  
Induced Apoptosis ◽  
Humoral Responses ◽  
Antigen Presenting

ABSTRACTModified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13Rby replacing the fragmented181R/182Rgenes of MVA with a functional anti-apoptotic gene,B13R, and confirmed its anti-apoptotic function against chemically induced apoptosisin vitro. In addition, MVA-B13Rshowed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13Rexpressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cellsin vitro. Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoringB13Rfunctionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cellsin vitroand augments vaccine-induced humoral immunity in mice.IMPORTANCEMVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes181R/182Rwith a functional version derived from vaccinia virus,B13R. Our results show that MVA-B13Rsignificantly delays apoptosis in antigen-presenting cells and muscle cellsin vitroand augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

scholarly journals Autophagy Promotes Duck Tembusu Virus Replication by Suppressing p62/SQSTM1-Mediated Innate Immune Responses In Vitro

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 22 ◽  
Author(s):  
Zhiqiang Hu ◽  
Yuhong Pan ◽  
Anchun Cheng ◽  
Xingcui Zhang ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Cell Model ◽  
Host Cells ◽  
Innate Immune Responses ◽  
Sequestosome 1 ◽  
Duck Tembusu Virus ◽  
Evolutionarily Conserved ◽  
New Strategy ◽  
Infected Cells ◽  
Tembusu Virus

Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-κB and IRF7 pathways, and changes in the NF-κB and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.

Sign in / Sign up

Export Citation Format

Share Document