Abstract Title

Translational Regulation and Expresion of HIV-1 Nef in the Central Nervous System

RAD Assignment Number

1607

Presenter Name

Kelly Wilson

Abstract

Significance: With over 36.7 million people infected today with Human Immunodeficiency Virus (HIV)and 2.1 million new infections each year, HIV presents a massive public health challenge. Introduction of antiretroviral therapies, such as cART, has led to increased life expectancy of HIV patients with a corresponding increase of AIDS induced neurological dysfunction known as HIV/NeuroAIDS. This is characterized by an increase in reactive astrocytes, with up to 20% of astrocytes becoming infected during severe cases. No specific treatment has been developed for HIV/NeuroAIDS, but It is known that astrocytes act as latent reservoirs for HIV-1. Understanding of this phenomenon is in need of expansion to improve treatment options.

Purpose: We sought to provide insight into this matter, by focusing on one particular HIV-1 protein, the Negative Regulatory Factor. Nef protein is one of three completely spliced HIV-1 proteins and has been implicated in HIV latency and mutations in Nef have been associated with long-term non progressive HIV infections. This coupled with astrocytes being a site of restricted HIV replication lead to the question of how Nef expression is regulated in astrocytes when compared to regulation in T lymphocytes, which have less restricted HIV replication.

Hypothesis: We hypothesized that translation of Nef is downregulated in astrocytes in the HIV infected central nervous system.

Materials and Methods: Nef transfected SVGA (human astrocyte cell line) and 293t (SV40 large T antigen-expressing human embryonic kidney cells) were used for in-vitro studies. Western blot, Real-time PCR, and Luciferase assay were utilized to analyze protein expression, Nef mRNA levels, and transcriptional activity, respectively. Additionally, expression of Nef in astrocytes was visualized in iNef mice (Inducible Nef transgenic mice) brain tissue with immunohistochemistry(IHC).

Results/Conclusions: Preliminary results show 293t cells expressing higher levels of Nef protein than SVGA cells. Analysis of mRNA levels, transcriptional activity, and IHC of iNef brain tissue are inconclusive. Further optimization of conditions is required to obtain consistent reproducible results.

Research Area

Microbiology/Infectious Disease

Presentation Type

Poster

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Translational Regulation and Expresion of HIV-1 Nef in the Central Nervous System

Significance: With over 36.7 million people infected today with Human Immunodeficiency Virus (HIV)and 2.1 million new infections each year, HIV presents a massive public health challenge. Introduction of antiretroviral therapies, such as cART, has led to increased life expectancy of HIV patients with a corresponding increase of AIDS induced neurological dysfunction known as HIV/NeuroAIDS. This is characterized by an increase in reactive astrocytes, with up to 20% of astrocytes becoming infected during severe cases. No specific treatment has been developed for HIV/NeuroAIDS, but It is known that astrocytes act as latent reservoirs for HIV-1. Understanding of this phenomenon is in need of expansion to improve treatment options.

Purpose: We sought to provide insight into this matter, by focusing on one particular HIV-1 protein, the Negative Regulatory Factor. Nef protein is one of three completely spliced HIV-1 proteins and has been implicated in HIV latency and mutations in Nef have been associated with long-term non progressive HIV infections. This coupled with astrocytes being a site of restricted HIV replication lead to the question of how Nef expression is regulated in astrocytes when compared to regulation in T lymphocytes, which have less restricted HIV replication.

Hypothesis: We hypothesized that translation of Nef is downregulated in astrocytes in the HIV infected central nervous system.

Materials and Methods: Nef transfected SVGA (human astrocyte cell line) and 293t (SV40 large T antigen-expressing human embryonic kidney cells) were used for in-vitro studies. Western blot, Real-time PCR, and Luciferase assay were utilized to analyze protein expression, Nef mRNA levels, and transcriptional activity, respectively. Additionally, expression of Nef in astrocytes was visualized in iNef mice (Inducible Nef transgenic mice) brain tissue with immunohistochemistry(IHC).

Results/Conclusions: Preliminary results show 293t cells expressing higher levels of Nef protein than SVGA cells. Analysis of mRNA levels, transcriptional activity, and IHC of iNef brain tissue are inconclusive. Further optimization of conditions is required to obtain consistent reproducible results.