UVM Theses and Dissertations
Format:
Print
Author:
Krementsov, Dimitry
Dept./Program:
Cell and Molecular Biology Program
Year:
2009
Degree:
Ph. D.
Abstract:
Human immunodeficiency virus (HIV) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). There are approximately 33 million people infected with HIV world-wide and this number continues to grow. Since HIV, like other retroviruses, possesses relatively few genes of its own, it relies heavily on the proteins of the host cell to replicate efficiently. Defining and understanding such cellular proteins may provide novel approaches to interfering with the life cycle of this virus. HIV vaccine efforts to date have failed, and it is unclear why this virus cannot be eliminated by the host immune system, despite a robust immune response. One potential explanation is the way this virus can spread very efficiently between infected and uninfected cells via so-called virological synapses (VSs). VSs are sites of transient adhesion between infected and uninfected cells, whereby virus is released in a polarized fashion into the synaptic cleft, greatly enhancing the efficiency of virus transfer. Further, such transfer of virus may be resistant to immune neutralization.
We and others showed that a group of cellular transmembrane proteins called the tetraspanins, including CD9, CD63 and CD81, exists in relatively discrete domains at the plasma membrane, and these domains are present at HIV-1 budding sites (including the VS). Thus, we tested whether these proteins were important factors in release and cell-to-cell transfer of HIV-1. Initially, we showed that treatment of HIV-1- producing cells with an anti-CD9 antibody reduced the efficiency of viral release. However, using knockdown and overexpression approaches, we showed that while expression of these proteins is not required for viral release, they appear to negatively regulate virus-induced membrane fusion, both at the virus-cell and cell-cell level. Overall, overexpression of these proteins, particularly CD63, decreased cell-free viral infectivity and productive cell-to-cell transmission of virus. Interestingly, knockdown of CD81 enhanced cell-to-cell transmission without increasing cell-free infectivity, suggesting another potential role for this tetraspanin in cell-to-cell transmission of virus. We also observed that these tetraspanins are downregulated in infected cells, suggesting the virus itself may modulate the levels of these proteins in order to achieve maximal replication.
Recently, we expanded on our previous studies which documented tetraspanin distribution relative to viral budding sites. Utilizing monovalent antibody fragments and GFP-fusion proteins, we analyzed the distribution these proteins in live cells and we found that these proteins are organized in less discrete (than previously reported) domains at the plasma membrane, but the expression of the HIV-1 Gag protein clusters them to budding sites in a time- and expression level-dependent manner. Overall, these studies define potential functions of a new group of cellular proteins, the tetraspanins, in the release and transmission of HIV-1 particles.
We and others showed that a group of cellular transmembrane proteins called the tetraspanins, including CD9, CD63 and CD81, exists in relatively discrete domains at the plasma membrane, and these domains are present at HIV-1 budding sites (including the VS). Thus, we tested whether these proteins were important factors in release and cell-to-cell transfer of HIV-1. Initially, we showed that treatment of HIV-1- producing cells with an anti-CD9 antibody reduced the efficiency of viral release. However, using knockdown and overexpression approaches, we showed that while expression of these proteins is not required for viral release, they appear to negatively regulate virus-induced membrane fusion, both at the virus-cell and cell-cell level. Overall, overexpression of these proteins, particularly CD63, decreased cell-free viral infectivity and productive cell-to-cell transmission of virus. Interestingly, knockdown of CD81 enhanced cell-to-cell transmission without increasing cell-free infectivity, suggesting another potential role for this tetraspanin in cell-to-cell transmission of virus. We also observed that these tetraspanins are downregulated in infected cells, suggesting the virus itself may modulate the levels of these proteins in order to achieve maximal replication.
Recently, we expanded on our previous studies which documented tetraspanin distribution relative to viral budding sites. Utilizing monovalent antibody fragments and GFP-fusion proteins, we analyzed the distribution these proteins in live cells and we found that these proteins are organized in less discrete (than previously reported) domains at the plasma membrane, but the expression of the HIV-1 Gag protein clusters them to budding sites in a time- and expression level-dependent manner. Overall, these studies define potential functions of a new group of cellular proteins, the tetraspanins, in the release and transmission of HIV-1 particles.