VACV Infection Research Articles

Visualizing cutaneous CD8+ T cells in multiple disease states to uncover common effector mechanisms

Abstract Cytotoxic CD8 +T cells are important for the control of many different pathologies in the skin, including cutaneous viral infections and skin cancers. One skin cancer, melanoma, is responsible for many deaths each year in the US alone. Despite this, we still have much to learn regarding the mechanisms that prevent and eliminate cutaneous cancers. Our laboratory has had a long-standing interest in better understanding the biology of T cells in the skin responding to the poxvirus vaccinia (VACV). To better understand global mechanisms of CD8+ T cell biology and effector function, we also established a melanoma model based on other published studies to compare with VACV infection. To do this, we adoptively transferred Rag2−/−Il2rg−/−mice with OT-I CD8+ T cells expressing dsRed, allowing us to easily visualize T cells using both frozen tissue sections and intravital microscopy. We then either 1) infected with VACV expressing or lacking cognate antigen (the SIINFEKL peptide) or 2) injected ovalbumin-expressing B16 melanoma cells into the outer ear pinnae. Primary analyses of this model via confocal microscopy of pinnae sections revealed OT-I T cells accumulating around the periphery of the melanoma lesion with a few cells infiltrating the tumor. Similarly, OT-I T cells accrued at the borders of foci of viral replication in the epidermis. Ongoing studies using this model will further characterize the immune response by visualizing the location of immune cells such as monocytes and neutrophils in relation to both tumors and virally infected cells. Comparisons between these two disease states may reveal shared mechanisms of interaction between effector CD8 +T cells and innate myeloid cells that traffic into the skin during pathology.

154 Nociceptor sensory neurons promote CD8 T cell response to VACV infection

154 Nociceptor sensory neurons promote CD8 T cell response to VACV infection

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling.

Receptor for activated C kinase 1 (RACK1) is a small ribosomal subunit protein that is phosphorylated by vaccinia virus (VacV) to maximize translation of postreplicative (PR) mRNAs that harbor 5' polyA leaders. However, RACK1 is a multifunctional protein that both controls translation directly and acts as a scaffold for signaling to and from the ribosome. This includes stress signaling that is activated by ribosome-associated quality control (RQC) and ribotoxic stress response (RSR) pathways. As VacV infection activates RQC and stress signaling, whether RACK1 influences viral protein synthesis through its effects on translation, signaling, or both remains unclear. Examining the effects of genetic knockout of RACK1 on the phosphorylation of key mitogenic and stress-related kinases, we reveal that loss of RACK1 specifically blunts the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at late stages of infection. However, RACK1 was not required for JNK recruitment to ribosomes, and unlike RACK1 knockout, JNK inhibitors had no effect on viral protein synthesis. Moreover, reduced JNK activity during infection in RACK1 knockout cells contrasted with the absolute requirement for RACK1 in RSR-induced JNK phosphorylation. Comparing the effects of RACK1 knockout alongside inhibitors of late stage replication, our data suggest that JNK activation is only indirectly affected by the absence of RACK1 due to reduced viral protein accumulation. Cumulatively, our findings in the context of infection add further support for a model whereby RACK1 plays a specific and direct role in controlling translation of PR viral mRNAs that is independent of its role in ribosome-based stress signaling. IMPORTANCE Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal protein that regulates translation directly and mediates signaling to and from the ribosome. While recent work has shown that RACK1 is phosphorylated by vaccinia virus (VacV) to stimulate translation of postreplicative viral mRNAs, whether RACK1 also contributes to VacV replication through its roles in ribosome-based stress signaling remains unclear. Here, we characterize the role of RACK1 in infected cells. In doing so, we find that RACK1 is essential for stress signal activation by ribotoxic stress responses but not by VacV infection. Moreover, although the loss of RACK1 reduces the level of stress-associated JNK activation in infected cells, this is an indirect consequence of RACK1's specific requirement for the synthesis of postreplicative viral proteins, the accumulation of which determines the level of cellular stress. Our findings reveal both the specific role of RACK1 and the complex downstream effects of its control of viral protein synthesis in the context of infection.

Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation

Abstract Tissue-resident memory (TRM) CD8+ T cells permanently reside within non-lymphoid tissues to provide a first-line of defense against invading pathogens. TRM T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation once they are recruited into non-lymphoid tissue microenvironments are largely undefined. To determine how T cell receptor (TCR) engagement promotes TRM formation, we utilized interferon-gamma (IFNγ)-YFP reporter T cells and found that only ~25% of the antigen-specific CD8+ T cells in the skin produced IFNγ during the course of a Vaccinia virus (VacV) infection. Strikingly, expression of IFNγ was strictly antigen-dependent and genome-wide transcriptional profiling revealed that IFNγ+ CD8+ T cells in the VacV-infected skin were becoming tissue-residents, suggesting that local antigen recognition identifies the dominant TRM precursor population. Mechanistically, TCR engagement caused effector CD8+ T cells to express Blimp1 and repress expression of the tissue egress receptor S1PR1 and the transcription factor KLF2, collectively preventing S1P-dependent migration. Interestingly, the S1PR1 antagonist CD69 was not transcriptionally upregulated by TCR stimulation. However, CD69 surface expression was Blimp1 dependent, suggesting that Blimp1-mediated repression of S1PR1 causes increased availability of CD69 protein to remain on the cell surface, a hallmark of TRM differentiation. Thus, antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor that promotes Blimp1 expression, ultimately causing stable expression of CD69 and retention of TRM CD8+ T cells in the skin following viral infection.

Temporal analysis of the plasma membrane proteome after vaccinia virus infection sheds light on virus strategies to evade the immune response

Abstract Vaccinia virus (VACV) is a poxvirus and the vaccine used to protect against variola virus and related orthopoxviruses. VACV encodes multiple proteins whose function is to evade the host immune response, contributing to an immune suppressive environment at the site of infection. There is, however, an incomplete understanding of how this immune suppression by VACV is consistent with a strong induction of immunological memory. There is especially little known about the relationship between NK cells and VACV, such as which ligands trigger NK activation and whether VACV uses strategies to interfere with the NK cell response. The purposes of this study is to clarify these open questions. Here, we performed a quantitative proteomic analysis of membrane-associated proteins from VACV-infected cells at various time points. Using tandem mass-tag spectrometry we detected about 70 viral proteins and 900 membrane-associated host proteins. Analysis revealed that VACV infection rapidly altered the expression of proteins with immune functions, downregulated the surface expression of plexins, ephrins, cadherins and selectively downregulated MHC-I proteins. Moreover, VACV prevented the upregulation of stress-induced ligands and death receptors. Finally, the kinetics of expression of VACV proteins at the surface of the infected cell were analysed. Collectively, these data constitute a valuable resource for the study of VACV and its interaction with the host immune response, provide insights into the interaction of NK cells with VACV, suggest novel VACV immune evasion mechanisms and shed light on which host factors are required to raise a protective immunological memory.

CD69 Targeting Enhances Anti-vaccinia Virus Immunity.

CD69 is highly expressed on the leukocyte surface upon viral infection, and its regulatory role in the vaccinia virus (VACV) immune response has been recently demonstrated using CD69-/- mice. Here, we show augmented control of VACV infection using the anti-human CD69 monoclonal antibody (MAb) 2.8 as both preventive and therapeutic treatment for mice expressing human CD69. This control was related to increased natural killer (NK) cell reactivity and increased numbers of cytokine-producing T and NK cells in the periphery. Moreover, similarly increased immunity and protection against VACV were reproduced over both long and short periods in anti-mouse CD69 MAb 2.2-treated immunocompetent wild-type (WT) mice and immunodeficient Rag2-/- CD69+/+ mice. This result was not due to synergy between infection and anti-CD69 treatment since, in the absence of infection, anti-human CD69 targeting induced immune activation, which was characterized by mobilization, proliferation, and enhanced survival of immune cells as well as marked production of several innate proinflammatory cytokines by immune cells. Additionally, we showed that the rapid leukocyte effect induced by anti-CD69 MAb treatment was dependent on mTOR signaling. These properties suggest the potential of CD69-targeted therapy as an antiviral adjuvant to prevent derived infections.IMPORTANCE In this study, we demonstrate the influence of human and mouse anti-CD69 therapies on the immune response to VACV infection. We report that targeting CD69 increases the leukocyte numbers in the secondary lymphoid organs during infection and improves the capacity to clear the viral infection. Targeting CD69 increases the numbers of gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing NK and T cells. In mice expressing human CD69, treatment with an anti-CD69 MAb produces increases in cytokine production, survival, and proliferation mediated in part by mTOR signaling. These results, together with the fact that we have mainly worked with a human-CD69 transgenic model, reveal CD69 as a treatment target to enhance vaccine protectiveness.

Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

SummaryVaccinia virus (VACV) has numerous immune evasion strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor κB (NF-κB), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics to quantify ∼9,000 cellular proteins and ∼80% of viral proteins at seven time points throughout VACV infection. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during infection. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a protein-based temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of HDAC5. Our approach thus identifies both a host antiviral factor and a viral mechanism of innate immune evasion.

Vaccinia virus E5 is a dominant inhibitor of the cytosolic DNA sensor cGAS

Abstract The cytosolic DNA sensor cGAS plays an important role in detecting viral nucleic acid, which leads to type I IFN production. We have previously shown that infection with conventional dendritic cells with modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia strain (VACV), induces IFN production via a cGAS/STING-dependent mechanism. However, MVA or VACV infection triggers cGAS degradation and its mechanism is still unknown. VACV is a cytoplasmic DNA virus, which encodes more than 200 genes. In this study, we screened 70 vaccinia viral early genes for inhibition of cGAS/STING pathway using a dual luciferase system. We found that vaccinia E5 is a dominant inhibitor of cGAS and is the key protein mediating cGAS degradation. MVAΔE5R induces much higher levels of type I IFN than MVA in multiple cell types, including bone marrow derived dendritic cells (BMDC), bone marrow-derived macrophages (BMDM), and skin primary fibroblasts. MVAΔE5R-mediated type I IFN production is dependent on cGAS. Furthermore, MVAΔE5R gains replication capability in cGAS−/− skin fibroblasts. As a vaccine vector, skin scarification or intradermal vaccination with MVAΔE5R-OVA leads to much higher OVA-specific CD8+ T cell responses than MVA-OVA in vivo. Intratumoral injection of MVAΔE5R leads to stronger anti-tumor immune responses and better survival compared with MVA. Finally, in an intranasal infection model, VACVΔE5R is at least 100-fold attenuated compared with WT VACV. Taken together, our results provide strong evidence that E5 is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production.

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.

RNAi-Mediated Depletion of Poxvirus Proteins.

RNA interference (RNAi) allows for transient, targeted depletion of cellular or viral proteins. Previously, small interfering RNA (siRNA) screens targeting cellular factors successfully identified several host genes that are required for VACV infection, and other viruses such as HIV. In this chapter, we outline how RNAi can be adapted to unravel the functions of poxvirus genes, using a 96-well format. Additionally, we describe two different high-throughput methods (flow cytometry and automated microscopy) to assess infection levels of an engineered VACV that encodes a fluorescent reporter protein under an early and/or late viral gene promoter.

Batf3-Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses against Respiratory Poxvirus Infection

Respiratory infection with vaccinia virus (VacV) elicits robust CD8+ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8+ effector T cell responses remains poorly defined. We used Batf3-/- mice to investigate the impact of CD103+ and CD8α+ dendritic cell (DC) deficiency on anti-VacV CD8+ T cell responses. We found that Batf3-/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+ T cells in the draining lymph nodes of Batf3-/- mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8+ T cells necessary for host resistance against acute respiratory VacV infection.IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8+ T cells play important role in resolving the primary infection. Effector CD8+ T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8+ T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8+ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

Vaccinia virus preferentially infects primary human memory B cells

Abstract Vaccinia virus (VACV) has shown an extreme preference for binding to and infection of primary human antigen-presenting cells including monocytes, macrophages, and dendritic cells in the peripheral blood. Although VACV infection in monocytes, macrophages, and dendritic cells has been examined, few studies have evaluated VACV infection in B cells. In this study, we characterized VACV binding to and infection of primary human B cells. Flow cytometric analysis revealed that B cells isolated from human peripheral blood mononuclear cells exhibited two distinct populations of B cells, referred to as Bsmall and Blarge. Phenotypic analysis demonstrated that the Bsmall compartment was more quiescent compared to the Blarge population and enriched with CD138-positive plasma cells. VACV preferentially entered into and infected memory B cells. Especially, VACV preferentially infected ex vivo CXCR5+ B cells, particularly CXCR5+HLA-DRhigh B cells. Assessment of VACV gene expression by RT-PCR in total B cells to evaluate permissiveness of VACV infection in B cells revealed early and intermediate viral gene expression, but little to no late viral gene expression, suggesting that VACV infection was aborted at the late viral gene expression stage in unstimulated B cells. Additionally, preliminary data demonstrated enhanced VACV binding to and infection of B cells stimulated by B cell receptor crosslinking and TLR-9 agonist treatment. With the risk of smallpox or monkeypox being used as bioterrorism agents and the multitude of adverse reactions to the current vaccine, understanding VACV infection in B cells is important for developing a better smallpox vaccine.

Vaccinia virus Western Reserve induces rapid surface expression of a host molecule detected by the antibody 4C7 that is distinct from CLEC2D.

In this study, the effect of active infection with vaccinia virus Western Reserve (VACV WR) on expression of C-type lectin domain family 2 (CLEC2D), a ligand of the human NK cell inhibitory receptor NKR-P1, was examined. As predicted, VACV infection led to a loss of CLEC2D mRNA in 221 cells, a B cell lymphoma line. Surprisingly, VACV infection of 221 cells caused a dramatic increase in cell surface staining for one CLEC2D-specific antibody, 4C7. There were no changes in other antibodies specific for CLEC2D and no indication that NK cells with NKR-P1A were inhibited, suggesting 4C7 detects a non-CLEC2D molecule following infection. The rapid increase in 4C7 signal requires virus attachment and is disrupted by UV treatment, but does not depend on new transcription or translation of either cellular or viral proteins. 4C7 does react with intracellular compartments, suggesting the molecule that is detected at the surface following infection is derived from an intracellular store. The phenomenon extends beyond lymphoid cells: it was observed in the non-human primate cell line Cos-7, but not with myxoma, a poxvirus distinct from VACV. To our knowledge, this is the first report of VACV or any poxvirus leading to rapid externalization of a host molecule. Among the VACV strains tested, the phenomenon was restricted to VACV WR and IHD-W, suggesting it has a virulence-, as opposed to a replication-related, function.

Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.

Tissue-resident memory (Trm) CD8(+) T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against reinfection. Whether local recognition of antigen in nonlymphoid tissues during infection can impact the formation of Trm populations remains unresolved. Using skin infections with vaccinia virus (VacV)-expressing model antigens, we found that local antigen recognition had a profound impact on Trm formation. Activated CD8(+) T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent, manner. However, after viral clearance, there was a subsequent ∼50-fold increase in Trm formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in nonlymphoid tissue caused CD8(+) T cells to rapidly express CD69 and be retained at the site of infection. Finally, Trm CD8(+) T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the nonlymphoid tissue microenvironment plays a critical role in the formation of functional Trm CD8(+) T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.

IL-15 stimulates core 2 O-glycan synthesis and regulates trafficking of memory CD8 T cells during viral skin infection

Abstract Following successful vaccination or pathogen clearance, recently activated CD8+ T cells that survive contraction differentiate into long-lived memory populations and provide host protection against re-infection. We have previously demonstrated that memory CD8+ T cells acquire the capacity to generate core 2 O-glycans in an antigen-independent manner, a post-translational modification required for binding to P- and E-selectin. In addition, we have shown that de novo synthesis of core 2 O-glycans can be regulated by IL-15 signaling and this modulates the ability for memory CD8+ T cells to traffic to inflamed tissues. Herein, we show that the trafficking of memory CD8+ T cells to Vaccinia Virus (VacV)-infected skin occurs in an antigen-independent manner, but that protective immunity requires antigen-specificity. Following epicutaneous infection with VacV, migratory dendritic cells express IL-15Rα, suggesting this cell type could trans-present IL-15 to T cells. In fact, nearly all memory CD8+ T cells in the draining lymph node during VacV infection synthesize core 2 O-glycans regardless of antigen specificity. IL-15 stimulates memory CD8+ T cells to specifically express ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (St3Gal4), an enzyme known to facilitate formation of sialyl Lewis X structures on core 2 O-glycans. Finally, we show that neutralizing IL-15 during VacV infection reduces E-selectin binding by circulating memory CD8+ T cells and results in decreased recruitment of these cells to the infected skin. Overall, these data identify IL-15 and core 2 O-glycan synthesis as critical regulators of memory CD8+ T cell trafficking to viral infection of the skin and perhaps other peripheral tissues.

Local antigen in non-lymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.

Abstract Tissue-resident memory (TRM) CD8+ T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against re-infection. One largely accepted paradigm is that activated CD8+ T cells are able to freely migrate to non-lymphoid tissues and differentiate into TRM independent of local antigen recognition. In contrast, using skin infections with Vaccinia virus (VacV) expressing model antigens, we found that local antigen recognition had a profound impact on TRM formation. Activated CD8+ T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent manner. However, following viral clearance, there was a subsequent ~50-fold increase in CD103+/CD69+ TRM formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in non-lymphoid tissue caused CD8+ T cells to rapidly express CD69 and be retained at the site of infection. Finally, TRM CD8+ T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the non-lymphoid tissue microenvironment plays a critical role in the formation of functional TRM CD8+ T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.

Cellular 5'-3' mRNA exonuclease Xrn1 controls double-stranded RNA accumulation and anti-viral responses.

SummaryBy accelerating global mRNA decay, many viruses impair host protein synthesis, limiting host defenses and stimulating virus mRNA translation. Vaccinia virus (VacV) encodes two decapping enzymes (D9, D10) that remove protective 5′ caps on mRNAs, presumably generating substrates for degradation by the host exonuclease Xrn1. Surprisingly, we find VacV infection of Xrn1-depleted cells inhibits protein synthesis, compromising virus growth. These effects are aggravated by D9 deficiency and dependent upon a virus transcription factor required for intermediate and late mRNA biogenesis. Considerable double-stranded RNA (dsRNA) accumulation in Xrn1-depleted cells is accompanied by activation of host dsRNA-responsive defenses controlled by PKR and 2′-5′ oligoadenylate synthetase (OAS), which respectively inactivate the translation initiation factor eIF2 and stimulate RNA cleavage by RNase L. This proceeds despite VacV-encoded PKR and RNase L antagonists being present. Moreover, Xrn1 depletion sensitizes uninfected cells to dsRNA treatment. Thus, Xrn1 is a cellular factor regulating dsRNA accumulation and dsRNA-responsive innate immune effectors.

Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.