Christopher M. Cherry and Dr. Gregory F. Burton, Chemistry and Biochemistry
HIV infects CD4 T cells through binding to CD4 and an appropriate co-receptor. Two co-receptors of concern in HIV biology are CXCR4 and CCR5 because of their affect on disease progression. The majority of transmittable HIV strains use CCR5 as a co-receptor. However, during the latter stages of disease the virus may shift from using CCR5 to using CXCR4 as its co-receptor. This change is referred to as a shift in tropism, and it is of major concern because it leads to rapid disease progression (3). The site and mechanism by which this shift occurs still elude scientists. Recent findings show that follicular dendritic cells (FDCs) in secondary lymphoid tissue up-regulate the expression of CXCR4 on CD4 T cells (6). In this study we show that germinal center T cells, which are located proximal to FDCs, are more readily infected by X4 tropic HIV even when simultaneously surrounded by a higher concentration of R5 virus. These results suggest that the germinal center is a unique environment that may facilitate a shift in the tropism of HIV.
Introduction
Human Immunodeficiency Virus (HIV) infects various cells of the immune system, but its primary target is the CD4 T cell. The bias of HIV to infect and replicate in CD4 T cells is known as its cell tropism. Infection is initiated when HIV binds to its primary receptor, CD4, a membrane bound protein on the surface of T cells. However, the presence of CD4 alone is not sufficient to allow HIV to enter the cell. There are several proteins that serve as co-receptors and facilitate the entrance of HIV into a CD4 T cell (1,2). Two of these co-receptors, CCR5 and CXCR4, are of major concern because of their effect on HIV pathogenesis. CCR5 is the primary co-receptor used by transmittable virus, while the use of CXCR4 only occurs, if it occurs at all, during the later stages of disease. This change in co-receptor use has been linked with rapid disease progression (3). The force behind this shift in HIV’s tropism is not understood and is thus the focus of many current research efforts.
The majority of HIV replication occurs within germinal centers (GC) of secondary lymphoid tissue (4-6). Germinal centers are unique immunological sites composed of follicular dendritic cells (FDCs), T cells, and B cells. The FDC and T cell are major players in HIV pathogenesis. Not only do FDCs act as long-term viral reservoirs (7), they also affect the characteristics of germinal center T cells – also referred to as CD57 T cells. CD57 T cells express high levels of the CXCR4 co-receptor in contrast to non-germinal center T cells. This up-regulation of CXCR4 is a result of the interaction between CD57 T cells and FDCs (8).
Given that GC T cells express high levels of CXCR4, we reasoned that these T cells would be preferentially infected by X4 tropic HIV. We report here that X4 tropic virus preferentially infect germinal center T cells, even when the T cells are incubated in the presence of X4 and R5 viruses at the same time.
Materials and Methods
GC T cell isolation
CD4+ CD57+ T cells were isolated from human tonsillar tissue. The tonsil was cut into small pieces and the cells were mechanically separated from tissue through repeat pipeting. Purification of these cells was performed by the process previously described (5).
HIV Infection and T cell DNA isolation
CD4+ CD57+ T cells were incubated for a period of six days in the presence of varying concentrations of both X4 and R5 viruses.
Proviral isolation and characterization
Nested set PCR was used to amplify a portion of the proviral env gene (V3 loop). PCR amplified products were ligated into the vector pCR3.1 (Invitrogen) at 15 C. STBL2 bacteria were transfected through heat shock (25 seconds at 42 C) and then plated. Individual colonies were selected and screened for the correct insert. The clones were amplified and plasma DNA was purified using QIAprep (Qiagen). The proviral gene was sequenced using the dye terminator method with previously developed primers.
Phylogenetic tree
The sequences were trimmed and a consensus sequence was made with sequencher (Gene Codes Corp. Inc.). The contig was aligned with Clustal X 1.81. The appropriate model of evolution was determined using Modeltest version 3.0626. A Neighbor-Joining tree was made using a maximum likelihood approach with PAUP*27.
Results
Germinal center T cells were incubated in the presence of both X4 and R5 tropic viruses. Several conditions were prepared – each one containing varying concentrations of X4 and R5 virus. Proviral DNA was isolated from the different conditions and the DNA was characterized to determine the tropism of the viruses that had successfully infected the GC T cells. The viral sequences were fit into a phylogenetic tree along with a control for both R5 and X4 viruses. We determined the tropism of the viruses that infected the GC T cells under the various conditions by observing where the viruses clustered on the phylogenetic tree. Analysis of figure 1 reveals that GC T cells are in fact preferentially infected by X4 Tropic HIV.
Conclusion
The data presented here clearly show that germinal center T cells are more readily infected by X4 tropic HIV even while they are predominantly surrounded by R5 tropic virus (fig. 1). These results are significant in that they give strength to the argument that the germinal center is a site that may facilitate the problematic shift in tropism of HIV. Further experiments are warranted and are currently being performed to investigate and either confirm or deny this possibility.
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