Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates

Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates

Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates
Herpes simplex virus (HSV) genomes are linear molecules (20) comprising covalently linked long (L) and short (S) segments, each containing unique sequences (UL and US) bracketed by inverted repeats (26). Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Using quantitative 3D colocalization analysis we show that upon coinfection of AAV and HSV the AAV replication protein Rep and ICP8 co‐reside in HSV replication domains. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA6 mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. In Res-M phi, HSV-1 DNA was present at early times post-infection but declined rapidly.

Single-stranded recombinant AAV DNA strongly stimulated this interaction, whereas double-stranded DNA was ineffective. Links to PubMed are also available for Selected References.

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