Reduced vacuolar ATPase protects mice from Friend virus infection - an unintended but instructive effect in Hif-2afl mice.

During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.

Immunization with a murine cytomegalovirus based vector encoding retrovirus envelope confers strong protection from Friend retrovirus challenge infection.

Immunization vectors based on cytomegalovirus (CMV) have attracted a lot of interest in recent years because of their high efficacy in the simian immunodeficiency virus (SIV) macaque model, which has been attributed to their ability to induce strong, unusually broad, and unconventionally restricted CD8+ T cell responses. To evaluate the ability of CMV-based vectors to mediate protection by other immune mechanisms, we evaluated a mouse CMV (MCMV)-based vector encoding Friend virus (FV) envelope (Env), which lacks any known CD8+ T cell epitopes, for its protective efficacy in the FV mouse model. When we immunized highly FV-susceptible mice with the Env-encoding MCMV vector (MCMV.env), we could detect high frequencies of Env-specific CD4+ T cells after a single immunization. While the control of an early FV challenge infection was highly variable, an FV infection applied later after immunization was tightly controlled by almost all immunized mice. Protection of mice correlated with their ability to mount a robust anamnestic neutralizing antibody response upon FV infection, but Env-specific CD4+ T cells also produced appreciable levels of interferon γ. Depletion and transfer experiments underlined the important role of antibodies for control of FV infection but also showed that while no Env-specific CD8+ T cells were induced by the MCMV.env vaccine, the presence of CD8+ T cells at the time of FV challenge was required. The immunity induced by MCMV.env immunization was long-lasting, but was restricted to MCMV naïve animals. Taken together, our results demonstrate a novel mode of action of a CMV-based vaccine for anti-retrovirus immunization that confers strong protection from retrovirus challenge, which is conferred by CD4+ T cells and antibodies.

Immunodominance of Adenovirus-Derived CD8+ T Cell Epitopes Interferes with the Induction of Transgene-Specific Immunity in Adenovirus-Based Immunization.

Adenovirus (Ad)-based immunization is a popular approach in vaccine development, and Ad-based vectors are renowned for their potential to induce strong CD8+ T cell responses to the encoded transgene. Surprisingly, we previously found in the mouse Friend retrovirus (FV) model that Ad-based immunization did not induce CD8+ T cell responses to the FV Leader-Gag-derived immunodominant epitope GagL85-93 We show now that induction of GagL85-93-specific CD8+ T cells was highly effective when leader-Gag was delivered by plasmid DNA immunization, implying a role for Ad-derived epitopes in mediating unresponsiveness. By immunizing with DNA constructs encoding strings of GagL85-93 and the two Ad-derived epitopes DNA-binding protein418-426 (DBP418-426) and hexon486-494, we confirmed that Ad epitopes prevent induction of GagL85-93-specific CD8+ T cells. Interestingly, while DBP418-426 did not interfere with GagL85-93-specific CD8+ T cell induction, the H-2Dd-restricted hexon486-494 suppressed the CD8+ T cell response to the H-2Db-restricted GagL85-93 strongly in H-2b/d mice but not in H-2b/b mice. This finding indicates that competition occurs at the level of responding CD8+ T cells, and we could indeed demonstrate that coimmunization with an interleukin 2 (IL-2)-encoding plasmid restored GagL85-93-specific CD8+ T cell responses to epitope strings in the presence of hexon486-494 IL-2 codelivery did not restore GagL85-93 responsiveness in Ad-based immunization, however, likely due to the presence of further epitopes in the Ad vector. Our findings show that seemingly immunodominant transgene epitopes can be dominated by Ad-derived epitopes. These findings underline the importance of thorough characterization of vaccine vectors, and modifications of vectors or immunogens may be required to prevent impaired transgene-specific immune responses.IMPORTANCE Ad-based vectors are widely used in experimental preclinical and clinical immunization studies against numerous infectious agents, such as human immunodeficiency virus, Ebola virus, Plasmodium falciparum, or Mycobacterium tuberculosis Preexisting immunity to Ad-based vectors is widely recognized as a hindrance to the widespread use of Ad-based vectors for immunizations in humans; however, our data show that an immune response to Ad-derived T cell epitopes can also result in loss or impairment of transgene-specific immune responses in prenaive vaccinees due to immune competition. Our results highlight that seemingly immunodominant epitopes may be affected by dominance of vector-derived epitopes, and modifications of the vector design or the immunogens employed in immunization may lead to more effective vaccines.

Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery.

BACKGROUND: In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines that were designed to induce either strong Friend virus GagL85-93-specific CD8+ T cell or antibody responses, respectively. To optimize the immunization outcome we evaluated vaccination strategies using combinations of these vaccines. RESULTS: While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine effectivity. We demonstrate that the co-immunization with GagL85-93/leader-gag encoding vectors together with envelope-encoding vectors abrogates the induction of GagL85-93-specific CD8+ T cells, and in successive immunization protocols the immunization with the GagL85-93/leader-gag encoding vector had to precede the immunization with an envelope encoding vector for the efficient induction of GagL85-93-specific CD8+ T cells. Importantly, the antibody response to envelope was in fact enhanced when the mice were adenovirus-experienced from a prior immunization, highlighting the expedience of this approach. CONCLUSIONS: To circumvent the immunosuppressive effect of envelope on immune responses to simultaneously or subsequently administered immunogens, we developed a two immunizations-based vaccination protocol that induces strong immune responses and confers robust protection of highly Friend virus-susceptible mice from a lethal Friend virus challenge.

Interference of retroviral envelope with vaccine-induced CD8+ T cell responses is relieved by co-administration of cytokine-encoding vectors.

BACKGROUND: Retroviral envelope (Env) proteins are known to exhibit immunosuppressive properties, which become apparent not only in retroviral infections, but also in gene-based immunizations using retroviral immunogens, where envelope interferes with the induction of CD8+ T cell responses towards another, simultaneously or subsequently delivered, immunogen. RESULTS: In the Friend retrovirus mouse model, immunization with a plasmid encoding the Friend murine leukemia virus (F-MuLV) Leader-Gag protein resulted in induction of a strong GagL85-93-specific CD8+ T cell response, while the response was completely abrogated by co-immunization with an F-MuLV Env-encoding plasmid. In order to overcome this interference of retroviral envelope, we employed plasmids encoding the cytokines interleukin (IL) 1ß, IL2, IL12, IL15, IL21, IL28A or granulocyte-macrophage colony-stimulating factor (GM-CSF) as genetic adjuvants. Co-application of plasmids encoding IL2, IL12, IL21, IL28A and especially GM-CSF rescued the induction of GagL85-93-specific CD8+ T cells in mice vaccinated with FV Leader-Gag and Env. Mice that were immunized with plasmids encoding Leader-Gag and Env and the cytokines IL1ß, IL12, IL15, IL28A or GM-CSF, but not Leader-Gag and Env without any cytokine, showed significantly reduced viral loads upon a high-dose Friend virus challenge infection. CONCLUSIONS: Our data demonstrate the potency of cytokine-encoding vectors as adjuvants and immune modulators in composite vaccines for anti-retroviral immunization.

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model.

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.

Tetherin promotes the innate and adaptive cell-mediated immune response against retrovirus infection in vivo.

Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 d postinfection with Friend retrovirus (FV) compared with mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, Friend retrovirus infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knockout mice at 2 wk postinfection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.

Murine leukemia virus Gag localizes to the uropod of migrating primary lymphocytes.

UNLABELLED: B and CD4(+) T lymphocytes are natural targets of murine leukemia virus (MLV). Migrating lymphocytes adopt a polarized morphology with a trailing edge designated the uropod. Here, we demonstrate that MLV Gag localizes to the uropod in polarized B cells and CD4(+) T cells. The uropod localization of MLV Gag was dependent on plasma membrane (PM) association and multimerization of Gag but independent of the viral glycoprotein Env. Basic residues in MA that are required for MLV Gag recruitment to virological synapses between HEK293 and XC cells were dispensable for uropod localization in migrating B cells. Ultrastructural studies indicated that both wild-type and basic-residue mutant Gag localized to the outer surface of the PM at the uropod. Late-domain mutant virus particles were seen at the uropod in form of budding-arrested intermediates. Finally, uropods mediated contact between MLV-infected B cells and uninfected T cells to form virological synapses. Our results suggest that MLV, not unlike HIV, accumulates at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod-mediated cell-cell contacts. IMPORTANCE: Viruses have evolved mechanisms to coordinate their assembly and budding with cell polarity to facilitate their spreading. In this study, we demonstrated that the viral determinants for MLV Gag to localize to the uropod in polarized B cells are distinct from the requirements to localize to virological synapses in transformed cell lines. Basic residues in MA that are required for the Gag localization to virological synapses between HEK293 and XC cells are dispensable for Gag localization to the uropod in primary B cells. Rather, plasma membrane association and capsid-driven multimerization of Gag are sufficient to drive MLV Gag to the uropod. MLV-laden uropods also mediate contacts between MLV-infected B cells and uninfected T cells to form virological synapses. Our results indicate that MLV accumulates at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod-mediated cell-cell contacts.

Experimental viral evolution to specific host MHC genotypes reveals fitness and virulence trade-offs in alternative MHC types.

The unprecedented genetic diversity found at vertebrate MHC (major histocompatibility complex) loci influences susceptibility to most infectious and autoimmune diseases. The evolutionary explanation for how these polymorphisms are maintained has been controversial. One leading explanation, antagonistic coevolution (also known as the Red Queen), postulates a never-ending molecular arms race where pathogens evolve to evade immune recognition by common MHC alleles, which in turn provides a selective advantage to hosts carrying rare MHC alleles. This cyclical process leads to negative frequency-dependent selection and promotes MHC diversity if two conditions are met: (i) pathogen adaptation must produce trade-offs that result in pathogen fitness being higher in familiar (i.e., host MHC genotype adapted to) vs. unfamiliar host MHC genotypes; and (ii) this adaptation must produce correlated patterns of virulence (i.e., disease severity). Here we test these fundamental assumptions using an experimental evolutionary approach (serial passage). We demonstrate rapid adaptation and virulence evolution of a mouse-specific retrovirus to its mammalian host across multiple MHC genotypes. Critically, this adaptive response results in trade-offs (i.e., antagonistic pleiotropy) between host MHC genotypes; both viral fitness and virulence is substantially higher in familiar versus unfamiliar MHC genotypes. These data are unique in experimentally confirming the requisite conditions of the antagonistic coevolution model of MHC evolution and providing quantification of fitness effects for pathogen and host. These data help explain the unprecedented diversity of MHC genes, including how disease-causing alleles are maintained.

Differential requirements of cellular and humoral immune responses for Fv2-associated resistance to erythroleukemia and for regulation of retrovirus-induced myeloid leukemia development.

To assess the possible contribution of host immune responses to the exertion of Fv2-associated resistance to Friend virus (FV)-induced disease development, we inoculated C57BL/6 (B6) mice that lacked various subsets of lymphocytes with FV containing no lactate dehydrogenase-elevating virus. Fv2(r) B6 mice lacking CD4(+) T cells developed early polycythemia and fatal erythroleukemia, while B6 mice lacking CD8(+) T cells remained resistant. Erythroid progenitor cells infected with spleen focus-forming virus (SFFV) were eliminated, and no polycythemia was observed in B cell-deficient B6 mice, but they later developed myeloid leukemia associated with oligoclonal integration of ecotropic Friend murine leukemia virus. Additional depletion of natural killer and/or CD8(+) T cells from B cell-deficient B6 mice resulted in the expansion of SFFV proviruses and the development of polycythemia, indicating that SFFV-infected erythroid cells are not only restricted in their growth but are actively eliminated in Fv2(r) mice through cellular immune responses.

Retrovirus glycoprotein functionality requires proper alignment of the ectodomain and the membrane-proximal cytoplasmic tail.

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the -1L, -2L, -3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5' and 3' splice sites.

The genome of the Friend murine leukemia virus (Fr-MLV) contains a 5' splice site (5'ss) located at 205 nt and a 3'ss located at 5489 nt. In our previous studies, it was shown that if the HindIII-BglII (879-1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr-MLV sequence, then cryptic splicing of env-mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII-BglII fragment were constructed. The vector, in which a 38 bp fragment (1612-1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183-1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI-NdeI (5140-5400 bp) fragment just upstream of the 3'ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140-5400 nt region located just upstream of the 3'ss is required for the splicing function of the 38 nt fragment and its flanking sequences.

Polyadenylation of Friend murine leukemia virus env-mRNA is affected by its splicing.

As splicing was previously found to be important for increasing Friend murine leukemia virus env-mRNA stability and translation, we investigated whether splicing of env-mRNA affected the poly(A) tail length using env expression vectors that yielded unspliced or spliced env-mRNA. Incomplete polyadenylation was detected in a fraction of the unspliced env-mRNA products in an env gene-dependent manner, showing that splicing of Friend murine leukemia virus plays an important role in the efficiency of complete polyadenylation of env-mRNA. These results suggested that the promotion of complete polyadenylation of env-mRNA by splicing might partially explain up-regulation of Env protein expression as a result of splicing.

Experimental viral evolution reveals major histocompatibility complex polymorphisms as the primary host factors controlling pathogen adaptation and virulence.

Using an experimental evolution approach, we recently demonstrated that the mouse-specific pathogen Friend virus (FV) complex adapted to specific major histocompatibility complex (MHC) genotypes, which resulted in fitness tradeoffs when viruses were exposed to hosts possessing novel MHC polymorphisms. Here we report the analysis of patterns of pathogen adaptation and virulence evolution from viruses adapting to one of three hosts that differ across the entire genome (A/WySn, DBA/2J and BALB/c). We found that serial passage of FV complex through these mouse genotypes resulted in significant increases in pathogen fitness (156-fold) and virulence (11-fold). Adaptive responses by post-passage viruses also resulted in host-genotype-specific patterns of adaptation. To evaluate the relative importance of MHC versus non-MHC polymorphisms as factors influencing pathogen adaptation and virulence, we compared the magnitude of fitness tradeoffs incurred by post-passage viruses when infecting hosts possessing either novel MHC polymorphisms alone or hosts possessing novel MHC and non-MHC polymorphisms. MHC polymorphisms alone accounted for 71% and 83% of the total observed reductions in viral fitness and virulence in unfamiliar host genotypes, respectively. Strikingly, these data suggest that genetic polymorphisms within the MHC, a gene region representing only -0.1% of the genome, are major host factors influencing pathogen adaptation and virulence evolution.

Self-renewal of leukemia stem cells in Friend virus-induced erythroleukemia requires proviral insertional activation of Spi1 and hedgehog signaling but not mutation of p53.

Friend virus induces erythroleukemia through a characteristic two-stage progression. The prevailing model proposes that during the initial, polyclonal stage of disease most of the infected cells terminally differentiate, resulting in acute erythrocytosis. In the late stage of disease, a clonal leukemia develops through the acquisition of new mutations--proviral insertional activation of Spi1/Pu.1 and mutation of p53. Previous work from our laboratory demonstrated that Friend virus activates the bone morphogenic protein 4 (BMP4)-dependent stress erythropoiesis pathway, which leads to the rapid expansion of stress erythroid progenitors, which are the targets for Friend virus in the spleen. We recently showed that stress erythroid progenitors have intrinsic self-renewal ability and therefore could function as leukemia stem cells (LSCs) when infected with Friend virus. Here, we show that the two stages of Friend virus-induced disease are caused by infection of distinct stress progenitor populations in the spleen. The development of leukemia relies on the ability of the virus to hijack the intrinsic self-renewal capability of stress erythroid progenitors leading to the generation of LSCs. Two signals are required for the self-renewal of Friend virus LSCs proviral insertional activation of Spi1/Pu.1 and Hedgehog-dependent signaling. Surprisingly, mutation of p53 is not observed in LSCs. These data establish a new model for Friend virus-induced erythroleukemia and demonstrate the utility of Friend virus as a model system to study LSC self-renewal.

The 0.3-kb fragment containing the R-U5-5'leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA.

BACKGROUND: A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5'leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood. RESULTS: Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5'splice site (5'ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5'ss. CONCLUSIONS: The 0.3-kb fragment containing the R-U5-5'leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing efficiency rather than transcription, nuclear export of spliced-mRNA, or poly (A) addition to mRNA. Seven nucleotides in the 0.3-kb fragment, which reside within the stem-loop structure that has been speculated to limit recognition of the 5'ss, could pinpoint the function of this region.

Kinetic studies of the effect of a 17-nucleotide difference in the 0.3-kb region containing the R-U5-5' leader sequence of Friend murine leukemia virus on viral gene expression.

In addition to the env gene, a 0.3-kb fragment containing the R-U5-5' leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr-MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3-kb fragment, and Rec5, carrying the 0.3-kb fragment of the non-neuropathogenic Fr-MLV clone 57. These analyses suggested that the 0.3-kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env-mRNA rather than the amount of total viral mRNA or viral production.

Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins (Envs). The envelope-mediated immunosuppression was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation specifically abolishes IS activity without affecting the "mechanical" fusogenic function of the entire envelope. Here, we genetically "switched off' the envelope-mediated immunosuppression of an infectious retrovirus, the Friend murine leukemia virus, while preserving mutant envelope infectivity both ex vivo and in vivo, thus allowing us to test the functional importance of envelope-mediated immunosuppression in retrovirus physiology. Remarkably, we show, in vivo, that the non-IS mutant virus displays the same propagation kinetics as its WT counterpart in irradiated immunocompromised mice but that it is rapidly and totally cleared from normal immunocompetent mice, which become fully protected against a challenge with the WT retrovirus. Using cell depletion strategies, we further establish that envelope-mediated immunosuppression enables the retrovirus to escape innate (natural killer cells) and adaptive (CD8 T cells) antiviral effectors. Finally, we show that inactivated mutant virions induce higher humoral and cellular responses than their WT counterparts. In conclusion, our work demonstrates the critical role of Env-induced immunosuppression for retrovirus propagation in vivo and identifies a unique definite target for antiretroviral therapies and vaccine strategies, also characterized in the human T-cell leukemia virus (HTLV) and xenotropic murine leukemia virus-related virus (XMRV) retroviruses, opening unprecedented prospects for the treatment of retroviral diseases.

Molecular breeding of viruses.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis.

The p53 tumour suppressor promotes cell-cycle arrest or apoptosis in response to cellular stress, such as DNA damage and oncogenesis. This role of p53 is important for its tumour-suppression function and depends, at least in part, on its ability to bind to specific DNA sequences and activate the transcription of target genes. The pathway through which p53 promotes apoptosis is not fully understood. Here we describe a new gene regulated by p53 that encodes a predicted protein of 915 amino acids in mice (910 amino acids in humans), which we have named Pidd. The mouse Pidd cDNA contains a p53 consensus DNA binding sequence upstream of the Pidd-coding region. This sequence element bound to p53 and conferred p53-dependent inducibility on a heterologous reporter gene. Moreover, Pidd RNA was induced by ionizing radiation in a p53-dependent manner and the basal level of Pidd RNA was dependent on Trp53 status. Overexpression of Pidd inhibited cell growth in a p53-like manner by inducing apoptosis. Antisense inhibition of Pidd expression attenuated p53-mediated apoptosis. Our data suggest that Pidd is an effector of p53-dependent apoptosis.