Epstein-Barr virus (EBV) is IntroductionEpstein-Barr virus (EBV)-associated lymphoid malignancies include a subset of Burkitt lymphoma, AIDS lymphoma, Hodgkin lymphoma, posttransplant lymphoma, age-associated B-cell lymphoma, and peripheral T-and NK-cell lymphoma. [1][2][3] The EBV life cycle includes distinct lytic and latent genetic programs. [4][5][6] In the lytic program, linear, double-stranded genomes are produced and packaged as virions that spread infection from cell to cell. In the latent program, only a few viral genes are transcribed, no viral progeny are produced, and infected cells are protected from apoptotic stimuli and in some circumstances driven to proliferate. The latent program predominates in tumors. There is increasing interest in pharmacologic activation of lytic viral gene expression in tumors. 7,8 Several therapeutic strategies requiring activation of EBV lytic genes for tumor cell killing have been described, 9,10 but concerns have been raised about the possible adverse effects of viral gene activation in patients treated with pharmacologic activators. 11,12 To better understand the drugs that induce EBV lytic gene expression, we screened a library of drugs approved by the Food and Drug Administration for human use and identified bortezomib as among the most potent. 10,13 Bortezomib is a proteasome inhibitor approved for use in the treatment of myeloma and mantle cell lymphoma. The cellular pathways that mediate the apoptotic effects of bortezomib in malignancy remain the focus of active investigation, but activation of the unfolded protein response (UPR) may play a critical role. [14][15][16] In the investigations described herein, we sought to better understand the pathways by which bortezomib may activate EBV lytic program gene expression. The screening assay that identified bortezomib as a lytic inducer focused on transcriptional activation of the promoter of the EBV ZTA gene (also referred to as BZLF1, ZEBRA, Z, or EB1). [17][18][19][20][21] ZTA is an immediate early transcriptional transactivator that induces other lytic genes and transcriptional trans-activators, resulting in virion production in permissive cell types. We were particularly interested in the possibility that CCAAT/enhancerbinding protein (C/EBP) family members might be important in ZTA promoter activation, because there is evidence that some C/EBP family members are polyubiquitinylated and degraded by the proteasome, 22,23 and also that C/EBP␣ and C/EBP␤ bind the ZTA promoter and activate EBV lytic program gene expression. [24][25][26][27] C/EBP family members share highly conserved C-terminal basic DNA-binding domains and basic leucine zipper domains (bZIPs). 26,27 As homo-or heterodimers, they modulate various cellular processes, including differentiation, metabolism, inflammation, proliferation, and malignancy. C/EBP␣ and C/EBP␤ each give rise to several different isoforms. The full-length isoforms of each protein include trans-activating and regulatory domains that can induce differentiation and inhibit prolif...

Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy studies showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with the microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a coinhibitor or alternate antiviral therapeutic agent in cases of resistance. IMPORTANCE Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious HIV. It was reported to inhibit herpesviruses in cell culture. Herpes simplex virus 1 (HSV-1) infections are common and often associated with several diseases.The studies we describe here confirm and extend earlier findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm) appear to be unaffected by NFV, whereas later steps (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell) are severely restricted by the drug. Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest that this drug may have applications for studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.

Key Points• ATRA and FLT3 TKIs have synergistic activity against FLT3/ITD 1 AML cell lines and patient samples.• Combination reduces the leukemia stem cell population and improves survival in genetic and xenograft AML mouse models.