Methods to inhibit or enhance the binding of viral DNA to genomic host DNA
a technology of viral dna and genomic host, which is applied in the field of methods to inhibit or enhance the binding of viral dna to genomic host dna, can solve problems such as interfering with host processes, and achieve the effect of eliminating the persistence of disease-causing viruses and great avidity
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experiment 1
[0134] Experiment 1
[0135] LANA and KSHV episomes localize similarly to host chromatin in KSHV infected cells. To determine if KSHV achieves latency by integrating to host DNA, we conducted fluorescent in-situ hybridization (FISH) studies with several viral cosmid probes on metaphase chromosomes prepared from a KSHV positive / EBV negative cell line (BC-3) derived from a body cavity lymphoma as well as a KSHV negative B cell line (BJAB). While viral DNA was not detected in the symmetrical pattern achieved by replication of a chromatid to which virus has integrated, it was consistently detected in a seemingly random association with host chromosomes (FIG. 1a). Furthermore, when similar chromosome spreads derived from the same KSHV positive cells were probed with a human serum that specifically recognizes LANA (FIG. 1b), we demonstrated that LANA localized to the host chromosomes in a pattern strikingly similar to that of KSHV specific DNA hybridization in FIG. 1a. FIG. 1c shows a non-sp...
experiment 2
[0136] LANA displays preferential binding to different regions of KSHV DNA in vitro. To ascertain if LANA had the capacity to bind specific sequences in KSHV DNA, .sup.32P-dCTP radiolabeled probes spanning the viral genome (FIG. 2b) were incubated with in vitro translated .sup.35S-methionine labeled LANA-myc fusion protein, followed by immunoprecipitation with anti-myc antibodies. To detect probes specifically bound to LANA, immunoprecipitates were quantified by liquid scintillation counting. Binding of LANA to DNA was expressed as a percentage of total probe that coimmunoprecipitated with LANA-myc. The results of this experiment, shown in FIG. 2a, demonstrated that LANA most preferentially bound a region of the KSHV genome referred to as Z2, located at approximately 127-140 kb on the right end of the viral genome (Russo, J. "Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)" PNAS 93:14862-14867, 1996). FIG. 2a is a representative of three separate experiments ...
experiment 3
[0137] LANA and KSHV DNA colocalize to metaphase chromosomes in KSHV infected cells. To determine if LANA colocalized with KSHV DNA, chromosome spreads were generated from cells prepared by fixing for only one hour in an effort to retain chromosome-associated antigens that would be lost by the typical overnight fixation used in standard FISH protocols as shown in FIG. 1a. These spreads were probed with KSHV DNA and amplified via a tyramide based fluorochrome deposition (NEN-Lifesciences) for increased sensitivity, followed immediately by anti-LANA immunofluorescence in an effort to colocalize viral DNA and LANA protein at host chromosomes. When both signals from the KSHV probe and the anti-LANA antibody were superimposed it became evident that both signals were colocalized to the chromosomes in BC-3 (FIG. 3d) but not BJAB cells (FIG. 3h).
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