Herpesvirus replication involves the expression of over 80 viral genes in a well ordered sequence, leading to the production of new virions. Viral genes expressed during the earliest phases of replication often regulate both viral and cellular genes. Therefore, they have the potential to bring about dramatic functional changes within the cell. Replication and transcription activator (RTA) is a potent immediate early transcription activator of the γ-herpesvirus family. This family includes Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, human pathogens associated with malignancy. Here we combine gene array technology with transcription factor profiling to identify the earliest DNA promoter and cellular transcription factor targets of RTA in the cellular genome. We find that expression of RTA leads to both activation and inhibition of distinct groups of cellular genes. The identity of the target genes suggests that RTA rapidly changes the cellular environment to counteract cell death pathways, support growth factor signaling, and also promote immune evasion of the infected cell. Transcription factor profiling of the target gene promoters highlighted distinct pathways involved in gene activation at specific time points. Most notable throughout was the high level of cAMP-response element-binding protein (CREB)-response elements in RTA target genes. We find that RTA can function as either an activator or an inhibitor of CREB-response genes, depending on the promoter context. The association with CREB also highlights a novel connection and coordination between viral and cellular “immediate early” responses.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The viral immediate early protein replication and transcription activator (RTA) plays a central role in mediating the balance between these two phases. Here, we demonstrate that a B cell terminal differentiation factor X-box binding protein 1 (XBP-1) can effectively initiates Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation by activating the RTA promoter, which results in the induction of other viral lytic transcripts. We also showed splicing of the XBP-1 mRNA which specifically occurs during B cell differentiation is critical in triggering KSHV reactivation. This work demonstrates the integration of KSHV reactivation mechanisms with host cell differentiation.

By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle.

Functional profiling technologies using arrayed collections of genome-scale siRNA and cDNA arrayed libraries enable the comprehensive global analysis of gene function. However, the current repertoire of high-throughput detection methodologies has limited the scope of cellular phenotypes that can be studied. In this report, we describe the systematic identification of mammalian growth-regulatory factors achieved through the integration of automated microscopy, pattern recognition analysis, and cell-based functional genomics. The effects of 7364 human and mouse proteins, encoded by individually arrayed cDNAs, upon proliferation and viability in U2OS osteosarcoma cells were evaluated in a live-cell, kinetic assay using quantitative image analysis. Overexpression of more than 86 cDNAs (1.15%) conferred dramatic increases in the proliferation, as determined cell enumeration. These included several known growth regulators, as well as previously uncharacterized ones (LRRK1, Ankrd25). In addition, novel functional roles for two genes (5033414D02Rik, 2810429O05Rik), now termed Gatp1 and Gatp2, respectively, were identified. Further analysis demonstrated that these encoded proteins promoted cellular proliferation and transformation in primary cells. Conversely, cells depleted for Gatp1 underwent apoptosis upon serum reduction, suggesting that Gatp1 is essential for cell survival under growth-factor-restricted conditions. Taken together, our findings offer new insight into the regulation of cellular growth and proliferation, and demonstrate the value and feasibility of assessing cellular phenotypes through genome-level computational image analysis.

Cellular homeoproteins have been shown to regulate the transcription of several viruses, including herpes simplex viruses, human papillomaviruses, and mouse mammary tumor viruses. Previous studies investigating the anti-viral mechanisms of several cyclin-dependent kinase inhibitors showed that the homeoproteins, pre B-cell leukemia transcription factor 1 (PBX1) and PBX-regulating protein-1 (PREP1), function as transcriptional activators of Moloney murine leukemia virus. Here, we examined the involvement of cellular homeoproteins in regulating the activity of the human cytomegalovirus immediate early (CMV IE) promoter. We identified a 45-bp element located at position -593 to -549 upstream of the transcription start site of the CMV IE gene, which contains multiple putative homeoprotein binding motifs. Gel shift assays demonstrated the physical association between a homeodomain protein, pancreatic-duodenal homeobox factor-1 (PDX1) and the 45-bp cytomegalovirus (CMV) region. We further determined that PDX1 represses the CMV IE promoter activity in 293 cells. Overexpression of PDX1 resulted in a decrease in transcription of the CMV IE gene. Conversely, blocking PDX1 protein synthesis and mutating the PDX1 binding sites enhanced CMV IE-dependent transcription. Collectively, our results represent the first work demonstrating that a cellular homeoprotein, PDX1, may be a repressor involved in regulation of human CMV gene expression.