engleski

Construction of an all-strain applicable construct for integration of DNA sequence into herpes simplex virus 1 genome – work in progress

Herpes simplex virus 1 (HSV-1) is a well-studied human pathogen involved in many ailments - from mild oral infections to serious health conditions such as keratitis and encephalitis. The replication of HSV-1 consists of a productive (lytic) and a latent phase (latency). During the lytic phase, the virus expresses its genes and generates viral progenies, inevitably destroying host cells. At the same time, the virus infects nearby neurons where active replication is halted and expression of genes is limited only to the region of the genome encoding latency associated transcripts (LAT). Importantly, the virus can reactivate from latency and cause recurrent diseases. The main goal of this study is to generate a simple construct for an efficient insertion of a sequence of interest into the HSV-1 genome using a cloned viral genome as a bacterial artificial chromosome (BAC) and homologous recombination in bacteria to study latency. We have generated a plasmid vector carrying homologous sequences to a region between UL3 and UL4 of the HSV-1 genome, a selection marker and a multiple cloning site (MCS) for cloning a sequence of interest (SOI). To evaluate and optimize the expression of the inserted SOI within the viral genome for different applications, we have generated several constructs carrying enhanced green fluorescent protein (EGFP) expressed under a strong promoter from cytomegalovirus (CMPp) or under an endogenous HSV-1 promoter. Also, in an effort to minimize the construct, we plan to test if a transcriptional termination signal cloned downstream of the SOI is required for the efficient expression of the inserted genes. The project and the current work in progress will be presented.

HSV-1, BAC mutagenesis

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