kinetic class
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2021 ◽  
Vol 95 (10) ◽  
Author(s):  
Annie Gravel ◽  
Wes Sanders ◽  
Éric Fournier ◽  
Arnaud Droit ◽  
Nathaniel Moorman ◽  
...  

ABSTRACT The “omics” revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T cells. Analyses were performed at different time points following infection, and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of the HHV-6B Z29 strain. The initial search focused on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts was observed in all samples analyzed. The presence of many of these new HHV-6B transcripts was confirmed by reverse transcriptase PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported open reading frames (ORFs), including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome. IMPORTANCE RNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48, and 72 h postinfection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names, to facilitate trace-back and correlate our work with previous studies on HHV-6B.



mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Shirley E. Braspenning ◽  
Tomohiko Sadaoka ◽  
Judith Breuer ◽  
Georges M. G. M. Verjans ◽  
Werner J. D. Ouwendijk ◽  
...  

ABSTRACT Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection. IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.



2007 ◽  
Vol 81 (24) ◽  
pp. 13761-13770 ◽  
Author(s):  
Amy H. Buck ◽  
Javier Santoyo-Lopez ◽  
Kevin A. Robertson ◽  
Diwakar S. Kumar ◽  
Martin Reczko ◽  
...  

ABSTRACT The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5′ splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.



2000 ◽  
Vol 74 (21) ◽  
pp. 9916-9927 ◽  
Author(s):  
S. W. Stingley ◽  
J. J. Garcia Ramirez ◽  
S. A. Aguilar ◽  
K. Simmen ◽  
R. M. Sandri-Goldin ◽  
...  

ABSTRACT More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early α27 (UL54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the α27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.



1998 ◽  
Vol 72 (3) ◽  
pp. 1910-1917 ◽  
Author(s):  
Suming Huang ◽  
Larry A. Hanson

To identify promoter regions that impart differential temporal regulation of channel catfish virus (CCV) genes, the transcriptional kinetics of an immediate-early gene and prospective early and late genes were characterized. A cDNA clone, designated IE3C, representing a third immediate-early transcript was identified. The 5′ end of the IE3C transcript was mapped to nucleotides 15,368 and 131,043 in the terminal repeat regions of the CCV genome. The full length of the transcript represented by the IE3C clone is 1,412 bp, and it most likely codes for the protein specified by open reading frame (ORF) 12. The putative product of ORF12 contains a consensus RING finger metal binding motif (C3HC4 structure). Temporal expression studies, in conjunction with protein synthesis and DNA replication inhibition, demonstrated that the IE3C transcript belongs to an immediate-early kinetic class, the ORF5 transcript is a member of the early kinetic class, and ORF39 and ORF46 are true late-kinetic-class genes. Additionally, we demonstrated that ORF38 transcription overlaps ORF39 and the products presumably share the same poly(A) signal. The 5′ ends of the transcripts encoding ORF38, ORF39, and ORF46 were mapped to nucleotides 44,862, 45,254, and 59,644, respectively, and potential transcriptional control elements were located.



Cell ◽  
1986 ◽  
Vol 46 (6) ◽  
pp. 865-872 ◽  
Author(s):  
A.P. Geballe ◽  
R.R. Spaete ◽  
E.S. Mocarski
Keyword(s):  


1978 ◽  
Vol 56 (5) ◽  
pp. 709-713 ◽  
Author(s):  
H. L. Chung ◽  
E. J. Bounsall

The base hydrolysis of trans-[Rh(cyclam)XY]+ (cyclam = 1,4,8,11-tetraazacyclotetradecane; X− and Y− = Cl−, Br−, and I−) are studied in aqueous solution over a range of OH− concentration and at various temperatures. The kinetics are done at a constant ionic strength with excess of [OH−] (smallest ratio = 200:1) so that pseudo first order rate constants are obtained for all the determinations. All of the reactions proceed with complete retention of configuration, and no trans-to-cis isomerization is found. The kinetic trans effect of these complexes is I− > Br− > Cl− on a rate basis, but based on ΔH≠, I− > Br− = Cl−. These Rh(III) complexes exhibit kinetic class (b) character on the ΔH≠ basis. The results of the rate constants and the activation parameters are interpreted in ternis of an SN1CB mechanism. The behavior of these complexes is compared to that of the other analogous complexes.



Author(s):  
Richard Kolodner ◽  
K.K. Tewari

Chloroplast (ct-) and mitochondrial (mt-) DNA's from pea, spinach, bean, and lettuce leaves have been studied for their molecular size and conformation. The ct-DNA's from these plants were found to band at a density of 1.698 ± 0.001 g/cm3 and denatured with a Tm of 84° ± 0.5°C. The corresponding mt-DNA's had a buoyant density of 1.706 ± 0.001 g/cm3 and denatured with a Tm of 88° ± 0.5°C. Both ct- and mt-DNA from these plants were found to have a homogeneous melting pattern. The sheared and denatured ctand mt-DNA's from these plants were found to renature as a single kinetic class with no indication of repeating sequences. The molecular weight of the ct-DNA from the higher plants studied has been found to be 95 ± 5 x 106 by renaturation rates assuming a value of 106 x 106 for T4 DNA.



Biochemistry ◽  
1970 ◽  
Vol 9 (7) ◽  
pp. 1547-1553 ◽  
Author(s):  
Stuart L. Laiken ◽  
Morton P. Printz


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