Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.

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The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC.

Local immunotherapy with an mRNA mixture encoding four cytokines mobilizes a systemic antitumor response and promotes tumor eradication.

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific singlestranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo.Implications: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.

Based on the observation that wild-type Kaposi's sarcoma-associated herpesvirus (KSHV) DNA can be detected in the oral cavity of healthy, immunocompetent individuals, we hypothesized that epithelial cells could be infected in vitro by wild-type (WT) KSHV isolated from immunocompetent individuals. Primary oral epithelial (P-EPI) cells and telomerase-immortalized oral epithelial cells were generated from human gingival tissue and were then infected in vitro with WT KSHV isolated from throat wash samples. Markers of lytic and latent KSHV infection were detected in cultures by 24 h postinfection by immunofluorescence confocal microscopic assays. The infectivity of the WT and BCBL virus was blocked by neutralizing antibodies against KSHV gB. The presence of KSHV DNA in these cells was confirmed by real-time PCR amplification of different regions of the viral genome. The significant in vitro viral replication that had occurred was inhibited by ganciclovir and by neutralizing antibodies against gB. When infected cultures were examined by scanning electron microscopy, thousands of KSHV particles were clearly visible across the surfaces of P-EPI cells. The detection of enveloped particles indicated that the infectious cycle had proceeded through assembly and egress. We thus demonstrated that oral WT KSHV isolated from immunocompetent individuals was able to infect and replicate in vitro in a relevant primary cell type. Furthermore, our results provide compelling evidence for KSHV transmission within infected oral epithelial cells derived from healthy, immunocompetent populations.Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 ) is the etiologic agent of Kaposi's sarcoma (KS) (12) and peripheral effusion lymphoma (PEL) (10). The human herpesviruses are generally ubiquitous among human populations, are generally shed in the oral cavity, and are primarily transmitted via the salivary route. However, current serological tests for KSHV have determined that the seroprevalence within the general U.S. population is low, suggesting that, unlike the other human herpesviruses (with the exception of herpes simplex virus type 2 [HSV-2]), KSHV is not ubiquitous among healthy human populations.The primary mode of KSHV transmission remains unresolved, as extensive evidence exists for both sexual (8,25,30,31) and oral (5, 29, 34) transmission routes in immunosuppressed individuals. However, recent epidemiology studies suggested that oral transmission of KSHV does occur among healthy populations (14,15). Complicating the transmission issue, the identities of the cell types harboring KSHV in vivo in the oral cavity and producing the virus detected in saliva are not known. Endothelial cells are the primary infected cells in KS lesions (7). Recently, KSHV antigens were detected in oral epithelial cells of an early KS lesion (44). In addition, the ability of the virus to infect primary human keratinocytes has also been demonstrated (3, 9). Based on the above evidence, we hypothesized that the wild-type (WT) isol...

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cellsto form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.The simplest infectious form of vaccinia virus (VACV), called the mature virion (MV), consists of an outer membrane with at least 20 associated proteins surrounding a core containing the double-stranded DNA genome, enzymes and factors required for the transcription of early genes, and numerous structural proteins (9, 19). During virus assembly, some MVs are wrapped with additional membranes that facilitate intracellular movement, exocytosis, and cell-to-cell spread (29). Extracellular enveloped virions (EVs) are essentially MVs with an additional membrane, which is opened or removed prior to the fusion of the MV and cell membrane during virus entry (18). Thus, the viral fusion proteins are components of the MV membrane rather than the EV-specific membrane (20). Mutagenesis and affinity purification studies have shown that at least eight MV membrane polypeptides are required for entry and have no other known function (4,16,21,22,25,26,28,30,31). The components of this entry/fusion complex (EFC) are conserved in all poxviruses, indicating a common mechanism of infection. Studies with VACV indicate that fusion may occur in a pH-independent manner at the plasma membrane (1, 7, 8) or in a pH-dependent manner within endosomes (32, 33).The EFC is also required for cell-cell fusion (28, 37), which can be induced by briefly lowering the pH (10, 13) or preventing expression of either the A56 (15, 24) or K2 (17, 34, 38) polypeptide. The glycosylated A56 polypeptide, referred to as the hemagglutinin (HA), is found on the plasma membrane of i...

Summary Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E-positive melanomas. It is therefore critical to prospectively identify all vemurafenib-resistance mechanisms prior to their emergence in the clinic. The vemurafenib-resistance mechanisms described to date do not result from secondary mutations within BRAFV600E. To search for possible mutations within BRAFV600E that can confer drug resistance, we developed a systematic experimental approach involving targeted saturation mutagenesis, selection of drug-resistant variants, and deep sequencing. We identified a single nucleotide substitution (T1514A, encoding L505H) that greatly increased drug resistance in cultured cells and mouse xenografts. The kinase activity of BRAFV600E/L505H was higher than that of BRAFV600E, resulting in cross-resistance to a MEK inhibitor. However, BRAFV600E/L505H was less resistant to several other BRAF inhibitors whose binding sites were further from L505 than that of PLX4720. Our results identify a novel vemurafenib-resistant mutant and provide insights into the treatment of melanomas bearing this mutation.