Relationship between retroviral DNA-integration-site selection and host cell transcription - PubMed (original) (raw)

Relationship between retroviral DNA-integration-site selection and host cell transcription

Lori F Maxfield et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Retroviral DNA integration occurs throughout the genome; however, local "hot spots" exist where a strong preference for certain sites over others are seen, and more global preferences associated with genes have been reported. Previous data from our laboratory suggested that there are fewer integration events into a DNA template when it is undergoing active transcription than when it is not. Because these data were generated by using a stably transfected foreign gene that was only weakly inducible, we have extended this observation by comparing integration events into a highly inducible endogenous gene under both induced and uninduced transcriptional states. To examine the influence of transcription on site selection directly, we analyzed the frequency and distribution of integration of avian retrovirus DNA into the metallothionein gene, before and after its induction to a highly sustained level of expression by addition of ZnSO4. We found a 6-fold reduction in integration events after 100-fold induction of transcription. This result implies that, despite an apparent preference for integration of retroviral DNA into transcribed regions of host DNA, high-level transcription can be inhibitory to the integration process. Several possible models for our observation are as follows. First, when a DNA template is undergoing active transcription, integration might be blocked by the RNA polymerase II complex because of steric hindrance. Alternatively, the integrase complex may require DNA to be in a double-stranded conformation, which would not be the case during active transcription. Last, transcription might lead to remodeling of chromatin into a structure that is less favorable for integration.

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Figures

Fig. 1.

Fig. 1.

Transcription of MT RNA with or without induction by zinc. QT6 cells were infected with the RAV-1 strain of ALV and either induced with 100 μM ZnSO4 or left uninduced. Total RNA was extracted at various times after infection and quantitated by real-time RT-PCR using SYBR Green fluorescence detection of the DNA product. (A) The quail MT gene showing primer pairs used to quantitate spliced and unspliced MT RNA by real-time RT-PCR. Arrows indicate the location of each primer. (B) Time course for induction of quail MT RNA as determined by the RT-PCR assay. The x axis represents time after induction by zinc, and the y axis represents fold increase in spliced or unspliced MT RNA relative to untreated cultures analyzed at the same time. Upper and Lower show the same data on two different time scales.

Fig. 2.

Fig. 2.

Effect of zinc induction on ALV DNA synthesis and integration. QT6 cells were infected with RAV-1 and either induced with 100 μM ZnSO4 or uninduced. Whole-cell DNA was extracted at the indicated times after infection. Integrated proviral DNA was quantitated by Southern blot hybridization using a probe specific for sequences within a highly conserved region of ALV pol. Arrows indicate the various forms of viral DNA. The bands representing the nonintegrated forms of ALV contain both 1-LTR and 2-LTR circles, because they were not distinguishable in this assay. Total DNA in the region of the gel (Genomic and integrated viral DNA) was visualized by staining with ethidium bromide. C33 cells are chicken embryo fibroblasts that contain a single endogenous provirus and provide a single-copy hybridization standard.

Fig. 3.

Fig. 3.

Effect of zinc induction on viral DNA integration. (A) Region within the quail MT gene where integration products were mapped in this assay system. Arrows indicate the positions of quail MT specific and viral LTR-specific PCR primers. The arrow marked with an asterisk indicates the location of the labeled primer-extension primer. (B) Representative data from one of six in vivo integration assays used to generate the histogram in Fig. 4. After amplification of eight parallel PCRs, each using the primers shown in A initiated with 20 μg of DNA from uninfected (Left), RAV-1 infected and uninduced (Center), or infected and Zinc-induced (Right) cells, specific amplification products were detected by extension of the 32P-end-labeled primer followed by electrophoresis in denaturing (sequencing) polyacrylamide gels. The sequencing ladder between the uninduced and induced lanes provided molecular size markers.

Fig. 4.

Fig. 4.

Location of integration events into the MT gene. PCR products generated in six separate experiments as described in Fig. 3 were used to determine the frequency of retroviral integration sites used at each base pair within the quail MT gene under both zinc-induction and uninduced conditions. The number of events observed at each site is plotted at the position of the site on the gene (Bottom). The arrow indicates the location within of the primer extension primer. The asterisk indicates the apparent use of the same site in both uninduced (twice) and zinc-induced cells.

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