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 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.

Functional and autoradiographic characterization of dopamine D2-like receptors in the guinea pig heart

2002 ◽  
Vol 80 (6) ◽  
pp. 578-587 ◽  
Author(s):  
María de Jesús Gómez ◽  
Guy Rousseau ◽  
Réginald Nadeau ◽  
Roberto Berra ◽  
Gonzalo Flores ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Western Blot ◽  
Dopamine Receptors ◽  
Negative Inotropic Effect ◽  
Cyclase Activity ◽  
Inotropic Effect ◽  
Receptor Subtypes ◽  
Guinea Pig Heart ◽  
Additional Information ◽  
The Right

Dopamine receptors include the D1- (D1 and D5 subtypes) and D2-like (D2, D3, and D4 subtypes) families. D1-like receptors are positively and D2-like receptors negatively coupled to the adenylyl cyclase. Dopamine D2-like (D4 subtype) receptors have been identified in human and rat hearts. However the presence of D2 and D3 receptor subtypes is unclear. Furthermore, their role in cardiac functions is unknown. By autoradiographic studies of guinea pig hearts, we identified D3 and D4 receptors, using the selective radioligands [3H]-7-OH-DPAT and [3H]emonapride (YM-09151-2 plus raclopride). Western blot analysis confirmed D3 and D4 receptors in the right and left ventricle of the same species. Selective agonists of D3 and D4 receptors (±)-7-OH-DPAT and PD 168 077 (10–9 to 10–5 M, respectively) induced a significant negative chronotropic and inotropic effect in the isolated guinea pig heart preparation. Negative inotropic effect induced by PD 168 077 was associated with an inhibition in cyclase activity. No changes in cyclase activity were found with (±)-7-OH-DPAT. The aim of this study is to support the presence of D3 and D4 receptors in the heart. Although our results suggest that D3 and D4 receptors are functionally active in the heart, we need additional information with an antagonist and an agonist of improved potency and selectivity to understand the respective roles of D3 and D4 receptors in the cardiac functions.Key words: Dopamine receptors (D2, D3, D4 subtypes), autoradiography, Western blot, cAMP, heart.

Morphometric analysis of the actions of angiotensin II on renal arterioles and glomeruli

1992 ◽  
Vol 262 (3) ◽  
pp. F367-F372 ◽  
Author(s):  
K. M. Denton ◽  
P. A. Fennessy ◽  
D. Alcorn ◽  
W. P. Anderson
Keyword(s):  
Electron Microscopy ◽  
Angiotensin Ii ◽  
Vascular Resistance ◽  
Morphometric Analysis ◽  
Unit Length ◽  
Average Difference ◽  
Outer Cortex ◽  
Efferent Arteriole ◽  
Transmission Electron ◽  
The Right

To study the effects of angiotensin II on afferent and efferent arteriole diameters and on intraglomerular dimensions, angiotensin II (20 ng.kg-1.min-1) or saline vehicle was infused intravenously for 20 min into anesthetized rabbits pretreated with enalapril. Both kidneys were perfusion fixed (glutaraldehyde), and vascular casts were made of the right kidneys using methacrylate. Morphometric analysis of the left kidneys using transmission electron microscopy revealed no significant effects of angiotensin II within the glomerulus, including the degree of mesangial contraction. The diameters of the afferent and efferent arteriole casts from the right kidneys were measured at 20, 50, and 75 microns from the glomerulus by scanning electron microscopy. In the outer cortex the mean diameters of the afferent and efferent arterioles were 14.1 +/- 0.8 and 9.7 +/- 0.5 microns, respectively, in the angiotensin II-infused rabbits, significantly less than in the control (vehicle) rabbits, 17.0 +/- 0.7 microns (P less than 0.001) and 10.7 +/- 0.4 microns (P less than 0.005), respectively. Calculation of the relative changes in vascular resistance, however, indicated that the effects of angiotensin II on efferent arteriole resistance (average difference 2.4 +/- 1.2 units/microns) were significantly greater per unit length than the effects on afferent arteriole resistance (average difference 0.9 +/- 0.3 units/microns). Thus infused angiotensin II caused greater reduction in afferent arteriolar diameter than in efferent, but the calculated increase in vascular resistance per micron was greater in efferent vessels due to their smaller resting diameter.

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