Current Research Summary
Questions being addressed by the Lingappa lab in 2011-2012:
1. Identifying cellular factors critical for HIV assembly, and understanding their mechanisms of action.
HIV-1 Gag proteins are synthesized in the cytoplasm but subsequently target to membranes where they assemble into spherical immature capsids that package the viral genome and undergo envelopment and release. Previous studies in our lab identified a pathway of assembly intermediates that culminates in formation of immature HIV-1 capsids (Lingappa 1997; Zimmerman 2002; Dooher 2004, Dooher 2007). These assembly intermediates are large ribonucleoprotein complexes that contain many cellular factors, most of which have not yet been identified. Our central hypothesis is that these assembly intermediates are sites of viral-host interactions critical for HIV capsid assembly and RNA packaging.
One of the proteins present in these identified a host protein present in HIV assembly intermediates, the cellular protein ABCE1 (Zimmerman 2002). A member of the ATP binding cassette superfamily (Kerr 2004), ABCE1 is highly conserved, present in all eukaryotes, and required for cell viability. In normal, uninfected cells, ABCE1 is critical for ribosome transport and assembly of the ribosome pre-initiation complex (see ABCE1 references). In cells expressing the HIV provirus, ABCE1 associates with assembly-competent HIV Gag polypeptides in high molecular mass complexes (Zimmerman 2002; Dooher 2004; Dooher 2007; Lingappa 2006). ABCE1 appears to be critical for progression of Gag through the HIV-1 capsid assembly pathway (Zimmerman 2002). Our current studies are focused on identifying additional cellular factors present in HIV assembly intermediates, and understanding how each of these host proteins functions during HIV assembly.
2. Understanding how HIV-1 Gag targets to membranes during HIV assembly.
During capsid assembly, Gag oligomerizes in the cytosol and then targets to the plasma membrane, where it undergoes further multimerization. Targeting of HIV Gag to the plasma membrane fails to occur in many rodent cells, leading to a block in virus assembly (reviewed in Swanson 2006). Our laboratory is interested in better understanding how trafficking of the critical membrane-targeting HIV assembly intermediate is regulated in human cells.
3. Determining how the HIV genome is selectively packaged during HIV assembly.
To produce an infectious virus, two copies of the HIV genome must be packaged into each nascent capsid during virus assembly. While the ratio of HIV genomic RNA to cellular RNA is low in the cytoplasm of infected cells where packaging takes place, this ratio is very high in released virus. Thus, viral genomic RNA is selectively recruited from the cytoplasm during assembly. HIV capsid assembly intermediates are likely to be the site of this selection process. Studies in our laboratory are in progress to determine when during the assembly pathway HIV genomic RNA first associates with the nascent HIV capsid.
4. Developing cell-free systems to understand virus capsid assembly and to identify novel virus assembly inhibitors.
Our group has developed cell-free systems for assembly of HIV-1 and other primate lentiviruses (Lingappa 1997; Dooher 2004; and Lingappa 2005), as well as hepatitis B virus (Lingappa 1994), hepatitis C virus ( Klein 2004; Klein 2005), and Venezuelan equine encephalitis virus (VEEV), which is an alphavirus on the list of NIAID priority pathogens (Kelley-Clarke, et al., submitted 2010). These cell-free capsid assembly systems have been used to identify assembly intermediates and understand viral-host interactions involved in assembly. Recently, Prosetta Bioconformatics adapted our VEEV screen for drugs that inhibit the assembly of alphaviruses. In collaboration with Prosetta Bioconformatics and USAMRIID, we are interested in understanding the mechanism of action of drugs identified in this screen.
5. Understanding how restriction factors are regulated in primary human cells.
A3G is one member of a family of cytidine deaminases. A3G acts as an antiviral "restriction factor" in human cells and is part of the "intrinsic immune system" (Sheehy 2002; Harris 2003). At least part of the antiviral effect of A3G results from A3G-mediated catastrophic hypermutation of the HIV genome. However, uncontrolled deaminase activity is also likely to be harmful to the cell. Little is known about how the activity of endogenously expressed APOBEC proteins is regulated in primary human cells. To address this question, our lab established a high throughput assay for measuring A3G enzymatic activity (Thielen 2007). Using this assay, we demonstrated that an RNase-insensitive inhibitor of A3G enzymatic activity is expressed in primary human T cells but not in the transformed cell lines that are frequently used to study restriction factors (Thielen 2007). Our data raise the possibility that this inhibitor limits the ability of A3G to catastrophically mutate the HIV genome. Currently, we are interested in determining the identity of this inhibitor, defining how regulation of this inhibitor affects HIV replication, and examining whether A3G deaminase activity can be modulated in primary human T cells. Unlike A3G, the cytidine deaminase APOBEC3A (A3A) is enzymatically active in primary human monocytes that have been treated with interferon alpha (Thielen 2010). The enzymatic activity of A3A in primary monocytes is modulated by other cytokines as well. The exact function of A3A in monocytes remains unclear, but it is likely to serve as a host defense against viral infection.