Enhanced Virus Spread Research Articles

A comprehensive analysis of the WRKY family in soybean and functional analysis of GmWRKY164-GmGSL7c in resistance to soybean mosaic virus

BackgroundSoybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear.ResultsHere, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element.ConclusionOur findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

Development of a novel, high-efficacy oncolytic herpes simplex virus type 1 platform equipped with two distinct retargeting modalities

To retarget oncolytic herpes simplex virus (oHSV) to cancer-specific antigens, we designed a novel, double-retargeted oHSV platform that uses single-chain antibodies (scFv) incorporated into both glycoprotein H (gH) and a bispecific adapter expressed from the viral genome to mediate infection predominantly via tumor-associated antigens. Successful retargeting was achieved using a nectin-1-detargeted HSV that remains capable of interacting with HVEM, the second canonical HSV entry receptor, and is therefore recognized by the adapter consisting of the virus-binding N-terminal 82 residues of HVEM fused to the target-specific scFv. We tested both an EpCAM- and a HER2-specific scFv separately and together to target cells expressing one, the other, or both receptors. Our results show not only dose-dependent, target receptor-specific infection in vitro, but also enhanced virus spread compared to single-retargeted virus. In addition, we observed effective infection and spreading of the EpCAM double-retargeted virus in vivo. Remarkably, a single intravenous dose of the EpCAM-specific virus eliminated all detectable tumors in a subcutaneous xenograft model, and the same intravenous dose appeared harmless in immunocompetent FVB/N mice. Our findings suggest that our double-retargeted oHSV platform can provide a potent and versatile, systemically deliverable class of anti-cancer therapeutics that specifically target cancer cells while ensuring safety.

Improved oncolytic activity of a reovirus mutant that displays enhanced virus spread due to reduced cell attachment

Wild-type reovirus serotype 3 Dearing (T3wt), a non-pathogenic intestinal virus, has shown promise as a cancer therapy in clinical trials, but it would benefit from an increased potency. Given that T3wt is naturally adapted to the intestinal environment (rather than tumors), we genetically modified reovirus to improve its infectivity in cancer cells. Various reovirus mutants were created, and their oncolytic potency was evaluated invitro using plaque size as a measure of virus fitness in cancer cells. Notably, Super Virus 5 (SV5), carrying five oncolytic mutations, displayed the largest plaques in breast cancer cells among the mutants tested, indicating the potential for enhancing oncolytic potency through the combination of mutations. Furthermore, in a HER2+ murine breast cancer model, mice treated with SV5 exhibited superior tumor reduction and increased survival compared with those treated with PBS or T3wt. Intriguingly, SV5 did not replicate faster than T3wt in cultured cells but demonstrated a farther spread relative to T3wt, attributed to its reduced attachment to cancer cells. These findings highlight the significance of increased virus spread as a crucial mechanism for improving oncolytic virus activity. Thus, genetic modifications of reovirus hold the potential for augmenting its efficacy in cancer therapy.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.

Novel Compound Inhibitors of HIV-1NL4-3 Vpu.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based ‘gain of function’ assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum

The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread.

Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread.

Abstract LB-206: The synergistic antitumor effect of the combination of three transgenes delivered by the oncolytic vaccinia virus, KLS-3020

Abstract Oncolytic viruses that selectively kill cancer cells are a promising platform for fighting cancer. Vaccinia virus is a good candidate for the oncolytic virus because it has several advantages such as a proven safety profile, broad host tropism, and larger capacity to harbor multiple transgenes. We removed three viral genes (C11R, K3L, and J2R) from wild-type vaccinia virus to obtain a backbone virus, which is selectively replicating in cancer cells. Then, we inserted three transgenes (PH20, sPD1-Fc, and IL-12) into the backbone virus to boost up the antitumor efficacy, which is the final construct named KLS-3020. We investigated the biological activity of transgenes with the intratumorally injected backbone viruses containing each single transgene in syngeneic mouse tumor models. At first, the backbone virus expressing PH20, an enzyme which can degrade the tumor extracellular matrix, showed enhanced virus spread compared to the empty backbone virus in melanoma or colon cancer. Likewise, locally expressed sPD1-Fc, which is a fusion protein of the extracellular domain of PD-1 and Fc region of an immunoglobulin, resulted in the increase of INF-γ+CD8+ and INF-γ+CD4+ T cells in melanoma, indicating T cell re-activation in the tumor site. Lastly, delivered IL-12 in lung cancer mouse model resulted in a significant increase of T-bet+CD4+ T cell populations, indicating the oriented differentiation of helper T cell into T helper 1 (Th1) cells. We compared the efficacy of oncolytic vaccinia virus containing a combination of three transgenes (KLS-3020) with ones expressing each single gene in syngeneic mouse tumor models and observed that KLS-3020 could control tumor growth more effectively than every backbone virus having a single gene. Finally, we evaluated the anti-tumor efficacy of KLS-3020 in various types of mouse tumor models. Intratumorally injected KLS-3020 could suppress the growth of various tumors without significant body weight loss. Our results suggest that enhanced virus spread combined with the immune-stimulatory activity of KLS-3020 synergistically increase antitumor efficacy in a broad range of tumor types. Citation Format: Jaeil Shin, Soon-Oh Hong, Minjung Kim, Heonsik Choi, Sujeong Kim. The synergistic antitumor effect of the combination of three transgenes delivered by the oncolytic vaccinia virus, KLS-3020 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-206.

Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis

SummaryIn sterile neuroinflammation, a pathological role is proposed for microglia, whereas in viral encephalitis, their function is not entirely clear. Many viruses exploit the odorant system and enter the CNS via the olfactory bulb (OB). Upon intranasal vesicular stomatitis virus instillation, we show an accumulation of activated microglia and monocytes in the OB. Depletion of microglia during encephalitis results in enhanced virus spread and increased lethality. Activation, proliferation, and accumulation of microglia are regulated by type I IFN receptor signaling of neurons and astrocytes, but not of microglia. Morphological analysis of myeloid cells shows that type I IFN receptor signaling of neurons has a stronger impact on the activation of myeloid cells than of astrocytes. Thus, in the infected CNS, the cross talk among neurons, astrocytes, and microglia is critical for full microglia activation and protection from lethal encephalitis.

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways.

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline-tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus-based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non-silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV-infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.

620. Cancer Associated Fibroblasts-Targeted Oncolytic Virus Results in Enhanced Antitumor Activity in Mouse Model

Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To target CAFs we developed a new oncolytic vaccinia virus that encodes a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the fibroblast activation protein (FAP) (FAP-T-cell engager-armed VV (FAP-TEA-VV)). FAP-TEA-VV induced robust FAP- and TRP2-specific T-cell responses in tumor environment, resulting in greater antitumor activity in the B16 melanoma model in comparison to unmodified VVs. FAP-TEA-VV enhanced tumor infiltration of CD8-positive T cells, and enhanced virus spread with tumors. Thus, targeting of CAFs has the potential to improve current virus therapy for cancer.

Exacerbation of Influenza Virus Infections in Mice by Intranasal Treatments and Implications for Evaluation of Antiviral Drugs

Compounds lacking oral activity may be delivered intranasally to treat influenza virus infections in mice. However, intranasal treatments greatly enhance the virulence of such virus infections. This can be partially compensated for by giving reduced virus challenge doses. These can be 100- to 1,000-fold lower than infections without such treatment and still cause equivalent mortality. We found that intranasal liquid treatments facilitate virus production (probably through enhanced virus spread) and that lung pneumonia was delayed by only 2 days relative to a 1,000-fold higher virus challenge dose not accompanied by intranasal treatments. In one study, zanamivir was 90 to 100% effective at 10 mg/kg/day by oral, intraperitoneal, and intramuscular routes against influenza A/California/04/2009 (H1N1) virus in mice. However, the same compound administered intranasally at 20 mg/kg/day for 5 days gave no protection from death although the time to death was significantly delayed. A related compound, Neu5Ac2en (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), was ineffective at 100 mg/kg/day. Intranasal zanamivir and Neu5Ac2en were 70 to 100% protective against influenza A/NWS/33 (H1N1) virus infections at 0.1 to 10 and 30 to 100 mg/kg/day, respectively. Somewhat more difficult to treat was A/Victoria/3/75 virus that required 10 mg/kg/day of zanamivir to achieve full protection. These results illustrate that treatment of influenza virus infections by the intranasal route requires consideration of both virus challenge dose and virus strain in order to avoid compromising the effectiveness of a potentially useful antiviral agent. In addition, the intranasal treatments were shown to facilitate virus replication and promote lung pathology.

Surf and Turf: Mechanism of Enhanced Virus Spread During Poxvirus Infection

Commentary on Doceul, V.; Hollinshead, M.; van der Linden, L.; Smith, G.L. Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science 2010, 327, 873–876.

Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread, and necrosis

To identify genes involved in resistance of tomato to Tomato yellow leaf curl virus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants where LeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.

Intratumoral Coadministration of Hyaluronidase Enzyme and Oncolytic Adenoviruses Enhances Virus Potency in Metastatic Tumor Models

Evaluate the codelivery of hyaluronidase enzyme with oncolytic adenoviruses to determine whether it improves the spread of the virus throughout tumors, thereby leading to a greater overall antitumor efficacy in tumor models. The optimal dose of hyaluronidase that provided best transduction efficiency and spread of a green fluorescent protein (GFP)-expressing adenovirus within tumors was combined with oncolytic viruses in tumor models to determine whether the combination treatment results in an improvement of antitumor efficacy. In mice injected with the adenovirus Ad5/35GFP and an optimal dose of hyaluronidase (50 U), a significant increase in the number of GFP-expressing cells was observed when compared with animals injected with virus only (P < 0.0001). When the oncolytic adenoviruses Ad5OV or Ad5/35 OV (OV-5 or OV5T35H) were codelivered with 50 U of hyaluronidase, a significant delay in tumor progression was observed, which translated into a significant increase in the mean survival time of tumor-bearing mice compared with either of the monotherapy-treated groups (P < 0.0001). Furthermore, the mice that received the combination of Ad5/35 OV and hyaluronidase showed the best antitumor efficacy. Importantly, the combination treatment did not increase the metastatic potential of the tumors. Lastly, the increase in virus potency observed in animals injected with both enzyme and virus correlated with enhanced virus spread throughout tumors. Antitumor activity and overall survival of mice bearing highly aggressive tumors are significantly improved by codelivery of oncolytic adenoviruses and hyaluronidase when compared with either of the monotherapy-treated groups, and it may prove to be a potent and novel approach to treating patients with cancer.

640. Oncolytic Transcriptionally Retargeted Adenovirus Expressing Nitroreductase in Combination with Antiangiogenic Drug RAD001

Top of pageAbstract We developed oncolytic adenoviruses that selectively target cancer cells with active |[beta]|-catenin signaling. To achieve this, Tcf promoters were inserted into early virus promoters E1A, E1B and E4. The virus (Ad5Tcf) demonstrated 1000-fold selectivity to tumor cells in vitro, but only a limited efficacy in vivo upon intravenous administration. We then used a range of approaches to increase in vivo efficacy of this virus. First, RGD motif was inserted into the fiber HI loop to improve infection of cancer cells. This modification increased overall efficacy of the virus and allowed infection of cell lines that did not express the natural CAR receptor. Next, we used combination therapy with m-TOR inhibitor, RAD001, which mainly acts as anti-angiogenic drug, to slow down tumor growth in a way that would not adversely affect the virus replication. This combined treatment with 10E11 particles of intravenous Ad5Tcf or Ad5Tcf-RGD virus and 5 mg/kg/day RAD001 was significantly better than any of the agents alone and could almost completely stop growth even of the most aggressive tumor xenografts. In addition, RAD001 is now undergoing phase II clinical trials as an immunosuppressant, and thus has a capability of limiting immune response to oncolytic adenoviruses. In our experiment, 5 mg/kg/day of RAD001 could completely abolish primary antibody response to adenovirus in immunocompetent mice. To further increase efficacy of the Tcf-retargeted virus, we expressed the Escherichia coli nitroreductase nfsB gene from an internal ribosome entry site (IRES) in the adenovirus L5 major late transcript. This enzyme converts prodrug CB1954 into a toxic derivative. The virus (Ad5Tcf-NTR) expressed nitroductase late with high specificity to cancer cells. This modification has markedly increased virus efficacy in vitro. In vivo, however, the gain from the enzyme-prodrug scheme was only marginal when used alone or in combination with RAD001. As we demonstrated, virus distribution in mouse xenografts was very uneven at late terms, and massively infected and dying tumor regions were separated by necrotic masses and connective tissue streaks that are impassable to the virus. Even minor isolated uninfected tumor fragments then quickly overgrew the killed part. As a conclusion, successful virus therapy of tumors would require further effort to improve virus delivery to achieve even infection and enhanced virus spread within the tumor.