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Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

ABSTRACT: We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

SUBMITTER: Stamey FR

PROVIDER: S-EPMC188616 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

Stamey F R FR Dominguez G G Black J B JB Dambaugh T R TR Pellett P E PE

Journal of virology 19950101 1

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated seque ...[more]

PMID: 7983761

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Project description: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 HHV-6B Z29 strain. The initial search focussed on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts were observed in all samples analyzed. The presence of many of these new HHV-6B transcripts were confirmed by RT-PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported ORFs, including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome.IMPORTANCERNA 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 hours post-infection. 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 in order to facilitate trace back and correlate our work with previous studies on HHV-6B.

Project description:BACKGROUND: Human herpesvirus-6 (HHV-6) is a beta-herpesvirus with 90% seroprevalence that infects and establishes latency in the central nervous system. Two HHV-6 variants are known: HHV-6A and HHV-6B. Active infection or reactivation of HHV-6 in the brain is associated with neurological disorders, including epilepsy, encephalitis, and multiple sclerosis. In a preliminary study, we found HHV-6B DNA in resected brain tissue from patients with mesial temporal lobe epilepsy (MTLE) and have localized viral antigen to glial fibrillary acidic protein (GFAP)-positive glia in the same brain sections. We sought, first, to determine the extent of HHV-6 infection in brain material resected from MTLE and non-MTLE patients; and second, to establish in vitro primary astrocyte cultures from freshly resected brain material and determine expression of glutamate transporters. METHODS AND FINDINGS: HHV-6B infection in astrocytes and brain specimens was investigated in resected brain material from MTLE and non-MTLE patients using PCR and immunofluorescence. HHV-6B viral DNA was detected by TaqMan PCR in brain resections from 11 of 16 (69%) additional patients with MTLE and from zero of seven (0%) additional patients without MTLE. All brain regions that tested positive by HHV-6B variant-specific TaqMan PCR were positive for viral DNA by nested PCR. Primary astrocytes were isolated and cultured from seven epilepsy brain resections and astrocyte purity was defined by GFAP reactivity. HHV-6 gp116/54/64 antigen was detected in primary cultured GFAP-positive astrocytes from resected tissue that was HHV-6 DNA positive-the first demonstration of an ex vivo HHV-6-infected astrocyte culture isolated from HHV-6-positive brain material. Previous work has shown that MTLE is related to glutamate transporter dysfunction. We infected astrocyte cultures in vitro with HHV-6 and found a marked decrease in glutamate transporter EAAT-2 expression. CONCLUSIONS: Overall, we have now detected HHV-6B in 15 of 24 patients with mesial temporal sclerosis/MTLE, in contrast to zero of 14 with other syndromes. Our results suggest a potential etiology and pathogenic mechanism for MTLE.

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Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

Publications

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

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