RESUMO
Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER) membrane into a functional N-terminal signal peptide (SP) and the C terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, and yet it was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was colocalized with Rab5-positive (Rab5+) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (containing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. IMPORTANCE Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Proteínas Nucleares/antagonistas & inibidores , Precursores de Proteínas/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.
Assuntos
Vetores Genéticos/metabolismo , Lentivirus/metabolismo , Biossíntese de Proteínas , Capuzes de RNA/metabolismo , DNA/metabolismo , Fator de Iniciação 4E em Eucariotos/biossíntese , Expressão Gênica , Interferons/metabolismo , RNA Interferente Pequeno/metabolismo , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.
Assuntos
Retrovirus Endógenos/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Superantígenos/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Animais , Proliferação de Células , Doença Crônica , Células Dendríticas/citologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Vírus do Tumor Mamário do Camundongo/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.
Assuntos
Vírus do Tumor Mamário do Camundongo/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Infecções por Retroviridae/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Animais , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Degradação Associada com o Retículo Endoplasmático , Humanos , Vírus do Tumor Mamário do Camundongo/química , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Infecções por Retroviridae/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.
Assuntos
Núcleo Celular/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/metabolismo , Transporte Ativo do Núcleo Celular , Processamento Alternativo , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Glicosilação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Microscopia de Fluorescência , Mutação , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Proteínas do Envelope Viral/genéticaRESUMO
Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We have shown that MMTV-encoded Rem protein inhibits proviral mutagenesis by the Apobec enzyme, activation-induced cytidine deaminase (AID) during viral replication in BALB/c mice. To further study the role of Rem in vivo , we have infected C57BL/6 (B6) mice with a superantigen-independent lymphomagenic strain of MMTV (TBLV-WT) or a mutant strain (TBLV-SD) that is defective in Rem and its cleavage product Rem-CT. Unlike MMTV, TBLV induced T-cell tumors in µMT mice, indicating that mature B cells, which express the highest AID levels, are not required for TBLV replication. Compared to BALB/c, B6 mice were more susceptible to TBLV infection and tumorigenesis. The lack of Rem expression accelerated B6 tumorigenesis at limiting doses compared to TBLV-WT in either wild-type B6 or AID-deficient mice. However, unlike proviruses from BALB/c mice, high-throughput sequencing indicated that proviral G-to-A or C-to-T changes did not significantly differ in the presence and absence of Rem expression. Ex vivo stimulation showed higher levels of mA3 relative to AID in B6 compared to BALB/c splenocytes, but effects of agonists differed in the two strains. RNA-Seq revealed increased transcripts related to growth factor and cytokine signaling in TBLV-SD-induced tumors relative to those from TBLV-WT, consistent with a third Rem function. Thus, Rem-mediated effects on tumorigenesis in B6 mice are independent of Apobec-mediated proviral hypermutation.
RESUMO
Studies of viral replication have provided critical insights into host processes, including protein trafficking and turnover. Mouse mammary tumor virus (MMTV) is a betaretrovirus that encodes a functional 98-amino-acid signal peptide (SP). MMTV SP is generated from both Rem and envelope precursor proteins by signal peptidase cleavage in the endoplasmic reticulum (ER) membrane. We previously showed that SP functions as a human immunodeficiency virus type 1 (HIV-1) Rev-like protein that is dependent on the AAA ATPase valosin-containing protein (VCP)/p97 to subvert ER-associated degradation (ERAD). SP contains a nuclear localization sequence (NLS)/nucleolar localization sequence (NoLS) within the N-terminal 45 amino acids. To directly determine the SP regions needed for membrane extraction and trafficking, we developed a quantitative retrotranslocation assay with biotin acceptor peptide (BAP)-tagged SP proteins. Use of alanine substitution mutants of BAP-tagged MMTV SP in retrotranslocation assays revealed that mutation of amino acids 57 and 58 (M57-58) interfered with ER membrane extraction, whereas adjacent mutations did not. The M57-58 mutant also showed reduced interaction with VCP/p97 in coimmunoprecipitation experiments. Using transfection and reporter assays to measure activity of BAP-tagged proteins, both M57-58 and an adjacent mutant (M59-61) were functionally defective compared to wild-type SP. Confocal microscopy revealed defects in SP nuclear trafficking and abnormal localization of both M57-58 and M59-61. Furthermore, purified glutathione S-transferase (GST)-tagged M57-58 and M59-61 demonstrated reduced ability to oligomerize compared to tagged wild-type SP. These experiments suggest that SP amino acids 57 and 58 are critical for VCP/p97 interaction and retrotranslocation, whereas residues 57 to 61 are critical for oligomerization and nuclear trafficking independent of the NLS/NoLS. Our results emphasize the complex host interactions with long signal peptides. IMPORTANCE Endoplasmic reticulum-associated degradation (ERAD) is a form of cellular protein quality control that is manipulated by viruses, including the betaretrovirus, mouse mammary tumor virus (MMTV). MMTV-encoded signal peptide (SP) has been shown to interact with an essential ERAD factor, VCP/p97 ATPase, to mediate its extraction from the ER membrane, also known as retrotranslocation, for RNA binding and nuclear function. In this paper, we developed a quantitative retrotranslocation assay that identified an SP substitution mutant, which is defective for VCP interaction as well as nuclear trafficking, oligomer formation, and function. An adjacent SP mutant was competent for retrotranslocation and VCP interaction but shared the other defects. Our results revealed the requirement for VCP during SP trafficking and the complex cellular pathways used by long signal peptides.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Sinais Direcionadores de Proteínas , Animais , Camundongos , Humanos , Proteína com Valosina/genética , Sinais Direcionadores de Proteínas/genética , Núcleo Celular/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Aminoácidos/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.
Assuntos
Neoplasias Mamárias Experimentais/imunologia , Vírus do Tumor Mamário do Camundongo/imunologia , Infecções por Retroviridae/imunologia , Superantígenos/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Feminino , Incidência , Glândulas Mamárias Animais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Baço/virologiaRESUMO
BACKGROUND: Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE). RESULTS: MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export. CONCLUSION: These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.
Assuntos
Expressão Gênica , Produtos do Gene rex/metabolismo , Vírus do Tumor Mamário do Camundongo/fisiologia , RNA Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linhagem Celular , Genes Reporter , Humanos , Camundongos , Ligação ProteicaRESUMO
Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland.
Assuntos
Diferenciação Celular , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Caseínas/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , DNA/metabolismo , Feminino , Deleção de Genes , Proteínas de Homeodomínio/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Proteínas Nucleares/genética , Gravidez , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/genética , Transcrição Gênica/genéticaRESUMO
Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes.IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.
Assuntos
Citidina Desaminase/antagonistas & inibidores , Vírus do Tumor Mamário do Camundongo/genética , Provírus/genética , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Feminino , Imunidade Inata , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Provírus/fisiologia , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Carga Viral/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação ViralRESUMO
Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.
Assuntos
Suscetibilidade a Doenças/microbiologia , Suscetibilidade a Doenças/virologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Vírus do Tumor Mamário do Camundongo/patogenicidade , Provírus/fisiologia , Vibrio cholerae/patogenicidade , Animais , Anticorpos Antibacterianos/fisiologia , Anticorpos Antivirais/fisiologia , Cólera/imunologia , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Provírus/genética , Ratos , Retroviridae/genética , Retroviridae/fisiologia , Infecções por Retroviridae/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/patogenicidade , Superantígenos/genética , Superantígenos/fisiologia , Transfecção , Infecções Tumorais por Vírus/imunologiaRESUMO
Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17-92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology.
Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/fisiologia , MicroRNAs/análise , Animais , Sequenciamento de Nucleotídeos em Larga Escala , CamundongosRESUMO
Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.IMPORTANCE Bacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction by retrotranslocation, which is part of a protein quality control system known as ER-associated degradation (ERAD) that is essential to cellular health. Through poorly understood mechanisms, certain pathogen-derived proteins are retrotranslocated but not degraded. We demonstrate here that MMTV SP retrotranslocation from the ER membrane avoids degradation through a unique process involving interaction with cellular p97 ATPase and failure to acquire cellular proteasome-targeting sequences.
Assuntos
Adenosina Trifosfatases/metabolismo , Evasão da Resposta Imune , Vírus do Tumor Mamário do Camundongo/imunologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Virais/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico , ProteóliseRESUMO
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. This review focuses on ERAD and its components as well as how viruses use this process to promote their replication and to avoid the immune response.
RESUMO
Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/química , Primers do DNA/química , Dimerização , Fibroblastos/metabolismo , Deleção de Genes , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Íntrons , Células Jurkat , Ligantes , Glândulas Mamárias Animais , Vírus do Tumor Mamário do Camundongo/genética , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Camundongos , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/metabolismo , Ratos , Receptores de Glucocorticoides/metabolismo , Receptores de Interleucina/metabolismo , Proteínas Recombinantes de Fusão/química , Retroviridae/genética , Ribonucleases/metabolismo , Frações Subcelulares , TransfecçãoRESUMO
Type B leukemogenic virus is a variant of mouse mammary tumor virus (MMTV) that causes thymic lymphomas rather than mammary tumors in mice. We demonstrate that conversion of a mammotropic MMTV to a T-cell-tropic virus requires two alterations in the long terminal repeat: (i) acquisition of a T-cell-specific enhancer and (ii) loss of transcriptional repression through deletion of negative regulatory elements (NREs) or by suppression of NRE activity after appropriate positioning of the enhancer.
Assuntos
Genes Reguladores , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/patogenicidade , Sequências Repetidas Terminais , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Humanos , Células Jurkat , Linfoma de Células T/virologia , Neoplasias Mamárias Experimentais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Provírus/genética , Provírus/patogenicidade , Infecções por Retroviridae/virologia , Linfócitos T/virologia , Transcrição Gênica , Transfecção , Infecções Tumorais por Vírus/virologiaRESUMO
Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus with two accessory genes, dut and sag. Cloned MMTV proviruses carrying a trimethoprim (trim) cassette in the envelope gene were defective for Gag protein production and the nuclear export of unspliced gag-pol RNA. Complementation experiments indicated that a trans-acting product was responsible for the Gag defect of such mutants. Analysis of MMTV-infected cells revealed the presence of a novel, doubly spliced RNA that encodes a putative product of 301 amino acids. Overexpression of cDNA from this RNA increased Gag levels from env mutant proviruses or reporter gene expression from unspliced mRNAs and allowed detection of a 33-kDa protein product, which has been named regulator of export of MMTV mRNA, or Rem. The Rem N terminus has motifs similar to the Rev-like export proteins of complex retroviruses, and mutation of the nuclear localization signal (NLS) abolished RNA export and detection within the nucleus. The Rem C terminus has few identifiable features, but removal of this domain increased Rem-mediated export, suggesting an autoregulatory function. A reporter vector developed from the 3' end of the MMTV provirus was Rem responsive and required both the presence of the MMTV env-U3 junction and a functional Crm1 pathway. The identification of a third accessory protein from a doubly spliced transcript suggests that MMTV is the first murine complex retrovirus to be documented. Manipulation of the MMTV genome may provide mouse models for human retroviral diseases, such as AIDS.
Assuntos
Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas Virais/metabolismo , Animais , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.