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1.  IL-2-independent and TNFα-dependent Expansion of Vβ5+ Natural Regulatory T Cells During Retrovirus Infection 
Friend virus (FV) infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of Neuropilin 1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4+ T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5+ Treg subset, which are known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5+ subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8+ T cell response rather than the level of FV infection. Surprisingly, the expansion and accumulation of the Vβ5+ Tregs was IL-2 independent but dependent upon TNFα. These experiments reveal a subset-specific Treg induction by a new pathway.
doi:10.4049/jimmunol.1202951
PMCID: PMC3739475  PMID: 23645880
2.  Interferon-alpha treatment inhibits acute Friend retrovirus replication primarily through the antiviral effector molecule Apobec3 
Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and HTLV-I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as ‘restriction factors’, which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. On the other hand, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1 and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.
doi:10.4049/jimmunol.1202920
PMCID: PMC3654153  PMID: 23315078
3.  Retroviral Immunology: Lessons from a mouse model 
Immunologic research  2009;43(0):160-166.
Friend virus is a murine retrovirus that causes acute disease leading to lethal erythroleukemia in most strains of mice. Strains of mice that mount the proper immune responses can recover from acute infection, but develop life-long chronic infections. The study of this infection has revealed the types of immune responses required for both recovery from the acute phase and the control of the chronic phase of infection. This knowledge has led to vaccines and therapeutics to prevent and treat infections and associated disease states. The FV model has provided insights into immunological mechanisms found to be relevant to human infections with viruses such as HIV-1 and HTLV-1.
doi:10.1007/s12026-008-8061-x
PMCID: PMC3708661  PMID: 18830572
Regulatory T cells; suppression; retrovirus; immunotherapy; CD8+ T cells; chronic viral infection; Friend virus
4.  CD4+ T Cells Develop Antiretroviral Cytotoxic Activity in the Absence of Regulatory T Cells and CD8+ T Cells 
Journal of Virology  2013;87(11):6306-6313.
Conventional CD4+ T cells play an important role in viral immunity. In most virus infections, they provide essential help for antiviral B and T cell responses. In chronic infections, including HIV infection, an expansion of regulatory T cells (Tregs) has been demonstrated, which can suppress virus-specific CD4+ T cell responses in vitro. However, the suppressive activity of Tregs on effector CD4+ T cells in retroviral infection is less well documented in vivo. We took advantage of a transgenic mouse in which Tregs can be selectively depleted to determine the influence of such cells on retrovirus-specific CD4+ T cell responses during an ongoing infection. Mice were infected with Friend retrovirus (FV), and Tregs were depleted during the acute phase of the infection. In nondepleted mice, activated CD4+ T cells produced Th1-type cytokines but did not exhibit any antiviral cytotoxicity as determined in a major histocompatibility complex (MHC) class II-restricted in vivo cytotoxic T lymphocyte (CTL) assay. Depletion of Tregs significantly increased the numbers of virus-specific CD4+ T cells and improved their cytokine production, whereas it induced only very little CD4+ T cell cytotoxicity. However, after dual depletion of Tregs and CD8+ T cells, conventional CD4+ T cells developed significant cytotoxic activity against FV epitope-labeled target cells in vivo and contributed to the control of virus replication. Thus, both Tregs and CD8+ T cells influence the cytotoxic activity of conventional CD4+ T cells during an acute retroviral infection.
doi:10.1128/JVI.00432-13
PMCID: PMC3648127  PMID: 23536666
5.  Negative Impact of IFNγ on Early Host Immune Responses to Retroviral Infection 
The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by co-infection with another microbe. It has been previously shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) co-infection. To investigate the mechanism of this impairment we examined LDV-induced innate immune responses and found LDV- specific induction of IFNα and IFNγ. LDV-induced IFNα had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFNγ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFNγ signaling indirectly modulated FV- specific CD8+ T cell responses. Second, intrinsic IFNγ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing antibody production. Results from this model reveal that IFNγ production can have detrimental impacts on early adaptive immune responses and virus control.
doi:10.4049/jimmunol.1201125
PMCID: PMC3424354  PMID: 22821964
6.  In vitro and in vivo Analyses of Regulatory T Cell Suppression of CD8+ T Cells 
The study of regulatory T cells (Treg) requires methods for both in vivo and in vitro analyses, both of which have different limitations, but which complement each other to give a more complete picture of physiological function. Our analyses have focused on Treg-mediated suppression of CD8+ T cells, and in particular Tregs induced by viral infection. One of the unique characteristics of virus-induced Tregs is that they can suppress in vitro without the requirement for additional stimulation. This ability correlates with their activated status in vivo. Furthermore, in vivo activated Tregs can suppress the function of CD8+ T cells both in vitro and in vivo, while leaving proliferation intact. Interestingly, further in vitro stimulation of these Tregs confers to them the ability to suppress both the function and proliferative ability of CD8+ T cell targets.
doi:10.1007/978-1-61737-979-6_4
PMCID: PMC3761880  PMID: 21287328
regulatory T cells; CD8+ T cells
8.  Virus-specific CD8+ T cells up-regulate PD-1 expression during acute Friend retrovirus infection but are highly cytotoxic and control virus replication 
It was recently reported that inhibitory molecules such as PD-1 were up-regulated on CD8+ T cells during acute Friend retrovirus infection, and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8+ T cells up-regulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1hi and PD-1lo subsets. More PD-1lo cells produced anti-viral cytokines such as IFNγ and TNFα, while more PD-1hi cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8+ T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus direct ex vivo analyses and in vivo experiments revealed high CD8+ T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.
doi:10.4049/jimmunol.1101612
PMCID: PMC3402334  PMID: 21873525
9.  A Single Nucleotide Polymorphism in Tetherin Promotes Retrovirus Restriction In Vivo 
PLoS Pathogens  2012;8(3):e1002596.
Tetherin is a membrane protein of unusual topology expressed from rodents to humans that accumulates enveloped virus particles on the surface of infected cells. However, whether this ‘tethering’ activity promotes or restricts retroviral spread during acute retrovirus infection in vivo is controversial. We report here the identification of a single nucleotide polymorphism in the Tetherin gene of NZW/LacJ (NZW) mice that mutated the canonical ATG start site to GTG. Translation of NZW Tetherin from downstream ATGs deleted a conserved dual-tyrosine endosomal sorting motif, resulting in higher cell surface expression and more potent inhibition of Friend retrovirus release compared to C57BL/6 (B6) Tetherin in vitro. Analysis of (B6×NZW)F1 hybrid mice revealed that increased Tetherin cell surface expression in NZW mice is a recessive trait in vivo. Using a classical genetic backcrossing approach, NZW Tetherin expression strongly correlated with decreased Friend retrovirus replication and pathogenesis. However, the protective effect of NZW Tetherin was not observed in the context of B6 Apobec3/Rfv3 resistance. These findings identify the first functional Tetherin polymorphism within a mammalian host, demonstrate that Tetherin cell surface expression is a key parameter for retroviral restriction, and suggest the existence of a restriction factor hierarchy to counteract pathogenic retrovirus infections in vivo.
Author Summary
Significant portions of the human and mouse genomes are comprised of retroviral sequences, revealing the long history of conflict between mammalian hosts and retroviruses that led to the evolution of host restriction factors. Nucleotide mutations in restriction factor genes provide a glimpse of this ongoing evolutionary process, but studies that directly probe the impact of restriction factor mutations during retrovirus infection are limited. In this study, we identified a single nucleotide mutation in the Tetherin host restriction gene that resulted in retention of Tetherin on the cell surface. In cell culture, Tetherin accumulates virions on the infected cell surface and prevents virion release, but some studies suggested that Tetherin might facilitate cell-to-cell virus spread. Our studies reveal that the Tetherin polymorphism inhibits retrovirus replication and disease. Thus, increased Tetherin cell surface expression enhanced the antiretroviral function of Tetherin. These results could have important implications in harnessing the biology of Tetherin for controlling pathogenic retroviruses such as HIV-1.
doi:10.1371/journal.ppat.1002596
PMCID: PMC3310811  PMID: 22457621
10.  Noninfectious Retrovirus Particles Drive the Apobec3/Rfv3 Dependent Neutralizing Antibody Response 
PLoS Pathogens  2011;7(10):e1002284.
Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.
Author Summary
Members of the APOBEC3 gene family can potently inhibit a broad range of retroviruses, including HIV-1. In cell culture, APOBEC3 counteracts retroviruses by: (1) reducing the infectivity of virions; and (2) inducing lethal G-to-A hypermutation in the next target cell. The selective advantage to the host of an ‘indirect’ restriction factor that is incorporated into virions and acts in the next target cell remains mysterious. We previously showed that Apobec3 encodes Rfv3, a classical resistance gene that controls the neutralizing antibody response against Friend retrovirus infection in mice. Here we demonstrate that Apobec3 promotes the release of substantial levels of noninfectious virions in the plasma during acute FV infection, resulting in a more potent antibody response directed against intact virions. Thus, we propose that APOBEC3 evolved as an innate mechanism to promote high concentrations of retrovirus antigen in a native but noninfectious form to effectively prime the neutralizing antibody response. These findings could have important implications for improving HIV-1 specific antibody responses.
doi:10.1371/journal.ppat.1002284
PMCID: PMC3188525  PMID: 21998583
11.  Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model 
Retrovirology  2011;8:76.
It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed.
doi:10.1186/1742-4690-8-76
PMCID: PMC3193819  PMID: 21943070
12.  Emergence of the GII-4 Norovirus Sydney2012 Strain in England, Winter 2012–2013 
PLoS ONE  2014;9(2):e88978.
Norovirus is the commonest cause of acute gastrointestinal disease and is the main aetiological agent of outbreaks of gastroenteritis, particularly in semi-closed environments. Norovirus infections in England typically peak between December and March each year. The most commonly detected norovirus strains belong to the genetically diverse genogroup-II genotype-4 (GII-4) genocluster and in the previous two norovirus winter seasons the majority of GII-4 strains in circulation worldwide have been genetically similar to the GII-4 strain New Orleans 1805/2009/USA. At the beginning of the 2012/13 season a genetically distinct GII-4 strain (Sydney 2012/NSW0514/2012/AU) was described which emerged worldwide during the winter of 2012/13. Here we describe the emergence of norovirus strains genetically related to Sydney2012 in England during the 2012/13 season to replace NewOrleans2009 strains as the most commonly detected variant of GII-4 norovirus in England. Furthermore, we demonstrate that whilst the emergence of Sydney2012 coincided with an early peak in the number of norovirus outbreaks, there was not an overall increase in norovirus activity compared to the previous season. Finally, we show that the Sydney2012 strain is associated with distinct genetic changes compared to the NewOrleans2009 strain, and these changes may have contributed to the emergence of the Sydney2012 strain.
doi:10.1371/journal.pone.0088978
PMCID: PMC3923861  PMID: 24551201
13.  Persistent Friend Virus Replication and Disease in Apobec3-Deficient Mice Expressing Functional B-Cell-Activating Factor Receptor ▿  
Journal of Virology  2010;85(1):189-199.
Rfv3 is an autosomal dominant gene that influences the recovery of resistant mice from Friend retrovirus (FV) infection by limiting viremia and promoting a more potent neutralizing antibody response. We previously reported that Rfv3 is encoded by Apobec3, an innate retrovirus restriction factor. However, it was recently suggested that the Rfv3 susceptible phenotype of high viremia at 28 days postinfection (dpi) was more dominantly controlled by the B-cell-activating factor receptor (BAFF-R), a gene that is linked to but located outside the genetically mapped region containing Rfv3. Although one prototypical Rfv3 susceptible mouse strain, A/WySn, indeed contains a dysfunctional BAFF-R, two other Rfv3 susceptible strains, BALB/c and A.BY, express functional BAFF-R genes, determined on the basis of genotyping and B-cell immunophenotyping. Furthermore, transcomplementation studies in (C57BL/6 [B6] × BALB/c)F1 and (B6 × A.BY)F1 mice revealed that the B6 Apobec3 gene significantly influences recovery from FV viremia, cellular infection, and disease at 28 dpi. Finally, the Rfv3 phenotypes of prototypic B6, A.BY, A/WySn, and BALB/c mouse strains correlate with reported Apobec3 mRNA expression levels. Overall, these findings argue against the generality of BAFF-R polymorphisms as a dominant mechanism to explain the Rfv3 recovery phenotype and further strengthen the evidence that Apobec3 encodes Rfv3.
doi:10.1128/JVI.01838-10
PMCID: PMC3014173  PMID: 20980520
14.  Complement Opsonization Enhances Friend Virus Infection of B Cells and Thereby Amplifies the Virus-Specific CD8+ T Cell Response ▿  
Journal of Virology  2010;85(2):1151-1155.
B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8+ T cells.
doi:10.1128/JVI.01821-10
PMCID: PMC3019994  PMID: 21047954
15.  The Glycosylated Gag Protein of a Murine Leukemia Virus Inhibits the Antiretroviral Function of APOBEC3▿  
Journal of Virology  2010;84(20):10933-10936.
APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.
doi:10.1128/JVI.01023-10
PMCID: PMC2950561  PMID: 20702647
16.  Innate retroviral restriction by Apobec3 promotes antibody affinity maturation in vivo1 
Apobec3/Rfv3 is an innate immune factor that promotes the neutralizing antibody response against Friend retrovirus (FV) in infected mice. Based on its evolutionary relationship to activation-induced deaminase (AID), Apobec3 might directly influence antibody class switching and affinity maturation independently of viral infection. Alternatively, the antiviral activity of Apobec3 may indirectly influence neutralizing antibody responses by reducing early FV-induced pathology in critical immune compartments. To distinguish between these possibilities, we immunized wild-type and Apobec3-deficient C57BL/6 (B6) mice with (4-hydroxy-3-nitrophenyl) acetyl (NP) hapten and evaluated the binding affinity of the resultant NP-specific antibodies. These studies revealed similar affinity maturation of NP-specific IgG1 antibodies between wild-type and Apobec3 deficient mice in the absence of FV infection. In contrast, hapten-specific antibody affinity maturation was significantly compromised in Apobec3-deficient mice infected with FV. In highly susceptible (B6 x A.BY)F1 mice, the B6 Apobec3 gene protected multiple cell types in the bone marrow and spleen from acute FV infection including erythroid, B, T and myeloid cells. In addition, B6 Apobec3 deficiency was associated with elevated immunoglobulin levels but decreased induction of splenic germinal center B cells and plasmablasts during acute FV infection. These data suggest that Apobec3 indirectly influences FV-specific neutralizing antibody responses by reducing virus-induced immune dysfunction. These findings raise the possibility that enabling Apobec3 activity during acute infection with human pathogenic retroviruses such as HIV-1 may similarly facilitate stronger virus-specific neutralizing antibody responses.
doi:10.4049/jimmunol.1001143
PMCID: PMC3024598  PMID: 20566830
17.  Tissue specific abundance of regulatory T cells correlates with CD8+ T cell dysfunction and chronic retrovirus loads 
Infection of mice with Friend virus induces the activation of CD4+ regulatory T cells (Tregs) that suppress virus-specific CD8+ T cells. This suppression leads to incomplete virus clearance and the establishment of virus persistence. We now show that Treg-mediated suppression of CD8+ T cells is tissue-specific, occurring in the spleen but not the liver. Regardless of infection status there was a five fold lower proportion of Tregs in the liver compared to the spleen, much lower absolute cell numbers, and the relatively few Tregs present expressed less CD25. Results indicated that reduced expression of CD25 on liver Tregs was due to microenvironmental factors including low levels of IL-2 production by CD4+ T helper cells in that tissue. Low CD25 expression on liver Tregs did not impair their ability to suppress CD8+ T cells in vitro. Correlating with the decreased proportion of Tregs in the liver was a significantly increased proportion of virus-specific CD8+ T cells compared to the spleen. Importantly, the virus-specific CD8+ T cells from the liver did not appear suppressed as they produced both IFNγ and granzyme B, and they also showed evidence of recent cytolytic activity (CD107a+). The functional phenotype of the virus-specific CD8+ T cells correlated with a ten-fold reduction of chronic Friend virus levels in the liver compared to the spleen. Thus, suppression of CD8+ T cells by virus-induced regulatory T cells occurs in a tissue-specific manner and correlates with profound effects on localized levels of chronic infection.
doi:10.4049/jimmunol.0900350
PMCID: PMC2775420  PMID: 19587016
18.  Effects of Acute and Chronic Murine Norovirus Infections on Immune Responses and Recovery from Friend Retrovirus Infection▿  
Journal of Virology  2009;83(24):13037-13041.
Murine norovirus (MNV) is a highly infectious but generally nonpathogenic agent that is commonly found in research mouse colonies in both North America and Europe. In the present study, the effects of acute and chronic infections with MNV on immune responses and recovery from concurrent Friend virus (FV) infections were investigated. No significant differences in T-cell or NK-cell responses, FV-neutralizing antibody responses, or long-term recovery from FV infection were observed. We conclude that concurrent MNV infections had no major impacts on FV infections.
doi:10.1128/JVI.01445-09
PMCID: PMC2786825  PMID: 19812147
19.  CD137-mediated immunotherapy for chronic viral infection 
Chronic viral infections cause high levels of morbidity and mortality worldwide making the development of effective therapies a high priority for improving human health. We have used mice infected with Friend virus (FV) as a model to study immunotherapeutic approaches to the cure of chronic retroviral infections. In chronic FV infections CD4+ T regulatory (Treg) cells suppress CD8+ T cell effector functions critical for virus clearance. Here we demonstrate that immunotherapy with a combination of agonistic anti-CD137 antibody and virus-specific, TCR transgenic CD8+ T cells produced greater than 99% reductions of virus levels within 2 weeks. In vitro studies indicated that the CD137-specific antibody rendered the CD8+ T cells resistant to Treg cell-mediated suppression with no direct effect on the suppressive function of the Treg cells. By two weeks post-transfer the adoptively transferred CD8+ T cells were lost, likely due to activation-induced cell death. The highly focused immunological pressure placed on the virus by the single specificity CD8+ T cells led to the appearance of escape variants indicating that broader epitope specificity will be required for long-term virus control. However, the results demonstrate a potent strategy to potentiate the function of CD8+ T cells in the context of immunosuppressive Treg cells.
PMCID: PMC2768524  PMID: 18390707
20.  Lactate Dehydrogenase-Elevating Virus Induces Systemic Lymphocyte Activation via TLR7-Dependent IFNα Responses by Plasmacytoid Dendritic Cells 
PLoS ONE  2009;4(7):e6105.
Background
Lactate dehydrogenase-elevating virus (LDV) is a natural infectious agent of mice. Like several other viruses, LDV causes widespread and very rapid but transient activation of both B cells and T cells in lymphoid tissues and the blood. The mechanism of this activation has not been fully described and is the focus of the current studies.
Principal Findings
A known inducer of early lymphocyte activation is IFNα, a cytokine strongly induced by LDV infection. Neutralization of IFNα in the plasma from infected mice ablated its ability to activate lymphocytes in vitro. Since the primary source of virus-induced IFNα in vivo is often plasmacytoid dendritic cells (pDC's), we depleted these cells prior to LDV infection and tested for lymphocyte activation. Depletion of pDC's in vivo eradicated both the LDV-induced IFNα response and lymphocyte activation. A primary receptor in pDC's for single stranded RNA viruses such as LDV is the toll-like receptor 7 (TLR7) pattern recognition receptor. Infection of TLR7-knockout mice revealed that both the IFNα response and lymphocyte activation were dependent on TLR7 signaling in vivo. Interestingly, virus levels in both TLR7 knockout mice and pDC-depleted mice were indistinguishable from controls indicating that LDV is largely resistant to the systemic IFNα response.
Conclusion
Results indicate that LDV-induced activation of lymphocytes is due to recognition of LDV nucleic acid by TLR7 pattern recognition receptors in pDC's that respond with a lymphocyte-inducing IFNα response.
doi:10.1371/journal.pone.0006105
PMCID: PMC2699471  PMID: 19568424
21.  Apobec3 encodes Rfv3, a gene influencing neutralizing antibody control of retrovirus infection 
Science (New York, N.Y.)  2008;321(5894):1343-1346.
Recovery from Friend Virus 3 (Rfv3) is a single autosomal gene encoding a resistance trait that influences retroviral neutralizing antibody responses and viremia. Despite extensive research for 30 years, the molecular identity of Rfv3 has remained elusive. Here we demonstrate that Rfv3 is encoded by Apobec3. Apobec3 maps to the same chromosome region as Rfv3 and has broad inhibitory activity against retroviruses including HIV. Not only did genetic inactivation of Apobec3 convert Rfv3-resistant mice to a susceptible phenotype, but Apobec3 was found to be naturally disabled by aberrant mRNA splicing in Rfv3-susceptible strains. The link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV and other human retroviral infections.
doi:10.1126/science.1161121
PMCID: PMC2701658  PMID: 18772436
22.  Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology 
PLoS ONE  2013;8(11):e79087.
DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.
doi:10.1371/journal.pone.0079087
PMCID: PMC3828344  PMID: 24244426
23.  Suppression of Acute Anti-Friend Virus CD8+ T-Cell Responses by Coinfection with Lactate Dehydrogenase-Elevating Virus▿  
Journal of Virology  2007;82(1):408-418.
Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8+ T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8+ T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8+ T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4+ T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.
doi:10.1128/JVI.01413-07
PMCID: PMC2224392  PMID: 17959678
24.  CD8+ T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection 
Journal of Virology  2005;79(16):10619-10626.
Virus-specific CD8+ T cells are critical for the control of acute Friend virus (FV) infections, but are rendered impotent by CD4+ regulatory T cells during the chronic phase of infection. The current study examines this CD8+ T-cell dysfunction by analyzing the production and release of cytolytic molecules by CD8+ T cells. CD8+ T cells with an activated phenotype (CD43+) from acutely infected mice produced all three key components of lytic granules: perforin, granzyme A, and granzyme B. Furthermore, they displayed evidence of recent degranulation and in vivo cytotoxicity. In contrast, activated CD8+ T cells from chronically infected mice were deficient in cytolytic molecules and showed little evidence of recent degranulation and poor in vivo cytotoxicity. Evidence from tetramer-positive CD8+ T cells with known virus specificity confirmed the findings from the activated subset of CD8+ T cells. Interestingly, perforin and granzyme A mRNA levels were not significantly reduced during chronic infection, indicating control at a posttranscriptional level. Granzyme B deficiency was associated with a significant decrease in mRNA levels, but posttranscriptional control also appeared to contribute to deficiency. These results demonstrate a broad impairment of cytotoxic CD8+ T-cell effector function during chronic retroviral infection and explain the inability of virus-specific CD8+ T cells to eliminate persistent virus.
doi:10.1128/JVI.79.16.10619-10626.2005
PMCID: PMC1182617  PMID: 16051854
25.  Alterations in Regulatory T Cells Induced by Specific Oligosaccharides Improve Vaccine Responsiveness in Mice 
PLoS ONE  2013;8(9):e75148.
Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet+ (Th1) activated CD69+CD4+ T cells (p<0.001) and reduced percentage of Gata-3+ (Th2) activated CD69+CD4+T cells (p<0.001) was detected in the mesenteric lymph nodes (MLN) of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4+Foxp3+) could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 + /T-bet+ (Th1-Tregs) was significantly reduced (p<0.05) in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.
In conclusion
These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.
doi:10.1371/journal.pone.0075148
PMCID: PMC3779252  PMID: 24073243

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