scholarly journals Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing

2016 ◽  
Vol 90 (15) ◽  
pp. 6989-6998 ◽  
Author(s):  
Craig J. Bierle ◽  
Kaitlyn M. Anderholm ◽  
Jian Ben Wang ◽  
Michael A. McVoy ◽  
Mark R. Schleiss
Keyword(s):  
Guinea Pig ◽  
Viral Genome ◽  
Genetic Manipulation ◽  
Spontaneous Mutation ◽  
Lytic Replication ◽  
Targeted Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Guinea Pig Cytomegalovirus ◽  
Infected Cells

ABSTRACTThe cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome inEscherichia coliand successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.IMPORTANCEThe cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infectionin vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

scholarly journals The Placental Response to Guinea Pig Cytomegalovirus Depends Upon the Timing of Maternal Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Zachary W. Berkebile ◽  
Dira S. Putri ◽  
Juan E. Abrahante ◽  
Davis M. Seelig ◽  
Mark R. Schleiss ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Digital Pcr ◽  
Maternal Infection ◽  
Junctional Zone ◽  
Viral Loads ◽  
Susceptibility To Infection ◽  
Increased Risk ◽  
Guinea Pig Cytomegalovirus ◽  
Infected Cells ◽  
Adverse Pregnancy

Human cytomegalovirus (HCMV) infects the placenta, and these placental infections can cause fetal injury and/or demise. The timing of maternal HCMV infection during pregnancy is a determinant of fetal outcomes, but how development affects the placenta’s susceptibility to infection, the likelihood of placental injury post-infection, and the frequency of transplacental HCMV transmission remains unclear. In this study, guinea pig cytomegalovirus (GPCMV) was used to model primary maternal infection and compare the effects of infection at two different times on the placenta. When guinea pigs were infected with GPCMV at either 21- or 35-days gestation (dGA), maternal and placental viral loads, as determined by droplet digital PCR, were not significantly affected by the timing of maternal infection. However, when the transcriptomes of gestational age-matched GPCMV-infected and control placentas were compared, significant infection-associated changes in gene expression were only observed after maternal infection at 35 dGA. Notably, transcripts associated with immune activation (e.g. Cxcl10, Ido1, Tgtp1, and Tlr8) were upregulated in the infected placenta. A GPCMV-specific in situ hybridization assay detected rare infected cells in the main placenta after maternal infection at either time, and maternal infection at 35 dGA also caused large areas of GPCMV-infected cells in the junctional zone. As GPCMV infection after mid-gestation is known to cause high rates of stillbirth and/or fetal growth restriction, our results suggest that the placenta becomes sensitized to infection-associated injury late in gestation, conferring an increased risk of adverse pregnancy outcomes after cytomegalovirus infection.

scholarly journals Dramatic Effects of 2-Bromo-5,6-Dichloro-1-β-d-Ribofuranosyl Benzimidazole Riboside on the Genome Structure, Packaging, and Egress of Guinea Pig Cytomegalovirus

2004 ◽  
Vol 78 (4) ◽  
pp. 1623-1635 ◽  
Author(s):  
Daniel E. Nixon ◽  
Michael A. McVoy
Keyword(s):  
Guinea Pig ◽  
Inhibitory Concentration ◽  
Unit Length ◽  
Genome Structure ◽  
Nuclease Digestion ◽  
Guinea Pig Cytomegalovirus ◽  
Infected Cells ◽  
Terminal Structure ◽  
The Right ◽  
Striking Contrast

ABSTRACT The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-β-d-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-β-d-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 μM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 μM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).

scholarly journals Herpesvirus BACs: Past, Present, and Future

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
Charles Warden ◽  
Qiyi Tang ◽  
Hua Zhu
Keyword(s):  
Vaccine Development ◽  
Genetic Manipulation ◽  
Large Family ◽  
Site Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Dna Viruses ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Viral Genes ◽  
Functional Analyses

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

scholarly journals HHi-FiVe: A high-fidelity genetic engineering pipeline for construction of herpesvirus-based vaccines

2021 ◽  
Author(s):  
Michael Jarvis ◽  
Thekla Mauch ◽  
Eleonore Ostermann ◽  
Yvonne Wezel ◽  
Jenna Nichols ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Genetic Manipulation ◽  
Rhesus Macaques ◽  
High Fidelity ◽  
Bacterial Artificial Chromosomes ◽  
Virus Challenge ◽  
Artificial Chromosomes ◽  
Vector Construction ◽  
Animal Populations ◽  
Cellular Tropism

Abstract Herpesvirus-based vectors are attractive for use both as conventional and as transmissible vaccines against emerging zoonoses in hard-to-reach animal populations. However, the threat of off-site mutations during genetic manipulation of vector genomes poses a significant challenge to vaccine construction. Herein, we present the HHi-FiVe (herpesvirus high-fidelity vector) construction pipeline for generating herpesvirus-based vectors by modifying bacterial artificial chromosomes (BACs) and monitoring integrity at each stage by complete genome sequencing. We used this pipeline to repair a highly mutated rhesus cytomegalovirus BAC containing an Ebola virus transgene. The vector derived from this BAC had been shown previously to protect rhesus macaques from lethal Ebola virus challenge by conventional vaccination. Repair of this BAC restored wild-type cellular tropism to the vector, which is essential for transmissible vaccination. Construction of this candidate transmissible vaccine against Ebola virus demonstrates the utility of the HHi-FiVe pipeline for creating precision-made herpesvirus-based vectors.

scholarly journals PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Maria Artesi ◽  
Vincent Hahaut ◽  
Basiel Cole ◽  
Laurens Lambrechts ◽  
Fereshteh Ashrafi ◽  
...  
Keyword(s):  
Viral Genome ◽  
Clonal Expansion ◽  
Endogenous Retroviruses ◽  
Viral Genomes ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Infected Cells ◽  
Insertion Sites ◽  
Viral Insertion ◽  
Hiv 1

AbstractThe integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

scholarly journals A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

2008 ◽  
Vol 82 (17) ◽  
pp. 8500-8508 ◽  
Author(s):  
Haiyan Li ◽  
Kazufumi Ikuta ◽  
John W. Sixbey ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Viral Genome ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

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