Prostate tumor cells infected with a recombinant influenza virus expressing a truncated NS1 protein activate cytolytic CD8+ cells to recognize noninfected tumor cells

Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.     

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

Negative regulation of the alpha interferon-induced antiviral response by the Ras/Raf/MEK pathway

Interferon (IFN) is one of the molecules released by virus-infected cells, resulting in the establishment of an antiviral state within infected and neighboring cells. IFN-induced antiviral response may be subject to modulation by the cellular signaling environment of host cells which impact the effectiveness of viral replication. Here, we show that cells with an activated Ras/Raf/MEK signaling cascade allow propagation of viruses in the presence of IFN. Ras-transformed (RasV12) and vector control NIH 3T3 cells were infected with vesicular stomatitis virus (VSV) or an IFN-sensitive vaccinia virus (delE3L) in the presence of alpha interferon. While IFN protected vector control cells from infection by both viruses, RasV12 cells were susceptible to viral infection regardless of the presence of IFN. IFN sensitivity was restored in RasV12 cells upon RNA interference (RNAi) knockdown of Ras. We further investigated which elements downstream of Ras are responsible for counteracting IFN-induced antiviral responses. A Ras effector domain mutant that can only stimulate the Raf kinase family of effectors was able to suppress the IFN response and allow VSV replication. IFN-induced antiviral mechanisms were also restored in RasV12 cells by treatment with a MEK inhibitor (U0126 or PD98059). Moreover, by using RNAi to MEK1 and MEK2, we determined that MEK2, rather than MEK1, is responsible for suppression of the IFN response. In conclusion, our results suggest that activation of the Ras/Raf/MEK pathway downregulates IFN-induced antiviral response.     

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.     

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-κB and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

Although infections with “natural” West Nile virus (WNV) and the chimeric W956IC WNV infectious clone virus produce comparable peak virus yields in type I interferon (IFN) response-deficient BHK cells, W956IC infection produces higher levels of “unprotected” viral RNA at early times after infection. Analysis of infections with these two viruses in IFN-competent cells showed that W956IC activated NF-κB, induced higher levels of IFN-β, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-β and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-β levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-κB activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification.     

The Ras-PI3K signaling pathway is involved in clathrin-independent endocytosis and the internalization of influenza viruses

Background

Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype.

Methodology/Principal Findings

Here, we identify the Ras–phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrin-independent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras–PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes.

Conclusions/Significance

Taken together, these results demonstrate that the Ras–PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.     

Herpes simplex virus disrupts NF-kappaB regulation by blocking its recruitment on the IkappaBalpha promoter and directing the factor on viral genes

Herpes simplex viruses (HSVs) are able to hijack the host-cell IkappaB kinase (IKK)/NF-kappaB pathway, which regulates critical cell functions from apoptosis to inflammatory responses; however, the molecular mechanisms involved and the outcome of the signaling dysregulation on the host-virus interaction are mostly unknown. Here we show that in human keratinocytes HSV-1 attains a sophisticated control of the IKK/NF-kappaB pathway, inducing two distinct temporally controlled waves of IKK activity and disrupting the NF-kappaB autoregulatory mechanism. Using chromatin immunoprecipitation we demonstrate that dysregulation of the NF-kappaB-response is mediated by a virus-induced block of NF-kappaB recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB-inhibitor. We also show that HSV-1 redirects NF-kappaB recruitment to the promoter of ICP0, an immediate-early viral gene with a key role in promoting virus replication. The results reveal a new level of control of cellular functions by invading viruses and suggest that persistent NF-kappaB activation in HSV-1-infected cells, rather than being a host response to the virus, may play a positive role in promoting efficient viral replication.     

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.     

Strategies of oncogenic microbes to deal with WW domain-containing oxidoreductase

WW domain-containing oxidoreductase (WWOX) is a well-documented tumor suppressor protein that controls growth, survival, and metastasis of malignant cells. To counteract WWOX’s suppressive effects, cancer cells have developed many strategies either to downregulate WWOX expression or to functionally inactivate WWOX. Relatively unknown is, in the context of those cancers associated with certain viruses or bacteria, how the oncogenic pathogens deal with WWOX. Here we review recent studies showing different strategies utilized by three cancer-associated pathogens. Helicobactor pylori reduces WWOX expression through promoter hypermethylation, an epigenetic mechanism also occurring in many other cancer cells. WWOX has a potential to block canonical NF-κB activation and tumorigenesis induced by Tax, an oncoprotein of human T-cell leukemia virus. Tax successfully overcomes the blockage by inhibiting WWOX expression through activation of the non-canonical NF-κB pathway. On the other hand, latent membrane protein 2A of Epstein–Barr virus physically interacts with WWOX and redirects its function to trigger a signaling pathway that upregulates matrix metalloproteinase 9 and cancer cell invasion. These reports may be just “the tip of the iceberg” regarding multiple interactions between WWOX and oncogenic microbes. Further studies in this direction should expand our understanding of infection-driven oncogenesis.     

“Armed” oncolytic herpes simplex viruses for brain tumor therapy

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be “armed” to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.Key words: oncolytic virus therapy, gene therapy, herpes simplex virus, viral vectors, G47Δ, G207, antitumor immunity