Codon-optimized and reporter-packaging strains of HIV-1
By: by Vesselin T. Tomov
Published: 2007
Language: eng
Number of pages: 177
Description: Currently there are an estimated 40.3 million people worldwide living with the human immunodeficiency virus (HIV). In the absence of a successful preventive or curative vaccine against this pathogen, treatment of HIV infection relies on antiretroviral drugs that target a limited number of rapidly mutating viral proteins. Here we present work on a novel strategy for vaccine development, aimed at creating a highly attenuated strain of HIV-1 through codon optimization. In addition, we describe viral reporter systems based on the incorporation of an exogenous enzyme directly into replication-competent virions, which were designed for compatibility with high-throughput screens of genetic libraries. Such screens should facilitate the discovery of novel antiviral targets of cellular origin. We have engineered a strain of HIV-1 whose protein-coding sequences conform to the codon usage of highly expressed human genes (SynHIV-1). This strategy was designed to take advantage of the facts that most organisms utilize synonymous codons in a highly biased way, and codon choice is a major determinant of gene expression. Based on available data regarding the effects of codon optimization on the processing of individual HIV-1 genes in human cells, we predicted that SynHIV-1 will replicate in a highly efficient and Rev-independent manner in cultured T lymphocytes. Our results indicate that, while individual codon-optimized genes are expressed efficiently in human cells, insertion of these genes into a live virus disrupts its replication. Furthermore, the inhibitory effects of codon-optimized sequences can be attributed directly to aberrant processing of the viral transcript by the cellular splicing machinery. A promising strategy for the discovery of new antiviral drug targets is the use of genetic screens to identify cellular proteins that affect viral replication.

Due to the magnitude of such experiments and the cumbersome nature of traditional HIV assays, large-scale screens for viral inhibitors typically rely on exogenous reporter systems. A number of such systems, based on the expression of non-secreted enzymes within target cells, have been described and used in automated, high-throughput screens of large collections of chemical compounds. However, these detection systems have two major disadvantages: (1) they require lysis of target cells in order to detect the signal, making it impossible to generate time-course data; and, more importantly, (2) they produce a readout even in the presence of late-stage viral antagonists, thus necessitating the prolonged growth of infected cultures before the effects of such inhibitors can be measured. Given the fact that it is currently expensive and impractical to automate the maintenance of cultured cells, the latter limitation makes assays based on exogenous reporters ill-suited for large-scale screens of genetic libraries, where the need to transfect genetic material into target cells prior to infection dictates suboptimal starting growth conditions. In an effort to create a viral detection system that would be compatible with automated HTS platforms involving genetic libraries, we have created reporter HIV-1 strains that incorporate luciferase derived from the copepod Gaussia princeps (GLUC) directly into replication-competent viruses via fusion to the virion-associated protein, Vpr. Alternatively, we have targeted GLUC to the viral envelope by expression as a glycophosphatidylinositol (GPI)-anchored protein in lipid rafts of reporter cell lines. These reporters are continuously packaged into virions without interfering with viral function, remain enzymatical active, and---in theory---could be used as true markers of productive HIV-1 replication after even a single round of infection.
Notes: Thesis (Ph.D., Division of Medical Sciences (Genetics))--Harvard University, 2007.

Includes bibliographical references.