Dynamics of KSHV gene expression during de novo infection and the role of LANA in immune modulation
AuthorThakker, Suhani Tushar
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Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human herpes virus that has been linked to the development of multiple malignancies including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Like all members of the herpesvirus family, KSHV establishes a lifelong persistence in the infected host. However, immediately upon infection, the virus has to overcome many challenges in the hostile cellular environment before it can establish a long-term infection. The remarkable success of the virus in establishing lifelong persistence in the infected host indicates that the virus is well equipped to manipulate the host environment very efficiently, even before it has a chance to fully express its genome. In order to understand how KSHV manipulates the host environment immediately upon infection, we must first understand which molecules are packaged in the virions and which viral genes are expressed during the initial time points following entry of the virus into the host cells. In chapter I, we probe these questions by performing a comprehensive analysis of the viral transcriptome in the purified KSHV virions. We also examine the dynamics of viral transcriptome at very early time-points following de novo infection of multiple cell lines permissive to long-term infection of KSHV. For this comprehensive study of viral transcriptome analysis, we used a high throughput approach of next-generation RNA sequencing. The results of this study identified many viral transcripts that are packaged into the virions and the transcripts that are actively transcribed during the initial infection period. Overall, the results of this study lay a foundation for future research targeting the viral genes whose expression may be critical for establishing a successful viral infection. One critical aspect of KSHV infection is its ability to persist throughout the life of the host. This can be attributed to its ability to efficiently hide from the host's immune surveillance. In chapter II, we identify a novel mechanism that helps the virus hide from the radar of host immunity. Our data sheds light on how one of the viral proteins, Latency- associated nuclear antigen (LANA), reduces the expression of major histocompatibility class II (MHC II) molecules, which are the molecules that are critical for reporting viral antigens to the immune cells of the host. We demonstrated that LANA binds with the proteins of regulatory factor X (RFX) complex, which are essential components of MHC II transcription machinery. The association of LANA with RFX complex reduces binding of the transcription factor called class II transactivator (CIITA) to the highly conserved promoters of MHC II genes. Binding of the CIITA to MHC II promoters is absolutely necessary for the expression of MHC II genes; by reducing the association of CIITA with the promoters of MHC II genes, LANA inhibits the expression of MHC II genes. KSHV infection is known to induce multiple pro-angiogenic cytokines that aid in extensive angiogenesis associated with Kaposi's sarcoma tumors. In chapter III, we demonstrate that LANA induces expression of a secreted angiogenic cytokine epidermal growth factor-like multiple 7 (EGFL7). Our data shows that LANA induces expression of EGFL7 in B cells, suggesting that it may act in a paracrine fashion during KSHV infection. The results of this research provide insight into an additional mechanism used by KSHV to promote angiogenesis. Targeting EGFL7 in combination with other important angiogenic cytokines induced during KSHV infection may increase the effectiveness of currently available anti-angiogenic therapy for Kaposi's sarcoma.