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1.
Proc Natl Acad Sci U S A ; 117(17): 9529-9536, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284399

RESUMO

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.


Assuntos
Quirópteros/virologia , Gammaretrovirus/isolamento & purificação , Animais , Austrália , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Phascolarctidae/virologia
2.
Arch Virol ; 165(1): 157-167, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31748876

RESUMO

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.


Assuntos
Gammaretrovirus/classificação , Técnicas de Genotipagem/métodos , Infecções por Retroviridae/epidemiologia , Animais , Animais Domésticos , Ásia , Cruzamento , Gatos , Gammaretrovirus/genética , Gammaretrovirus/isolamento & purificação , Noruega , Filogenia , Filogeografia , Infecções por Retroviridae/virologia , Espanha , Tanzânia
3.
Arch Virol ; 164(11): 2735-2745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31486907

RESUMO

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.


Assuntos
Retrovirus Endógenos/genética , Gammaretrovirus/patogenicidade , Phascolarctidae/virologia , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/veterinária , Animais , Animais de Zoológico/virologia , Linhagem Celular , Coinfecção/veterinária , Coinfecção/virologia , Retrovirus Endógenos/classificação , Retrovirus Endógenos/isolamento & purificação , Feminino , Gammaretrovirus/classificação , Gammaretrovirus/genética , Gammaretrovirus/isolamento & purificação , Japão/epidemiologia , Masculino , Provírus/genética , Infecções por Retroviridae/virologia , Tupaia/virologia , Carga Viral
4.
Virology ; 535: 154-161, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31302509

RESUMO

Most viruses infect only a few hosts, but the xenotropic and polytropic mouse leukemia viruses (X/P-MLVs) are broadly infectious in mammalian species. X/P-MLVs use the XPR1 receptor for cell entry, and tropism differences are due to polymorphisms in XPR1 and the viral envelope. To characterize these receptor variants and identify blocks to cross-species transmission, we examined the XPR1 receptors in six mammalian species that restrict different subsets of X/P-MLVs. These restrictive receptors have replacement mutations in regions implicated in receptor function, and some entry restrictions can be relieved by glycosylation inhibitors. Mutation of the cow and hamster XPR1 genes identified a shared, previously unrecognized receptor-critical site. This G/Q503N replacement dramatically improves receptor function. While this substitution introduces an N-linked glycosylation site, XPR1 receptors are not glycosylated indicating that this replacement alters the virus-receptor interface independently of glycosylation. Our data also suggest that an unidentified glycosylated cofactor may influence X/P-MLV entry.


Assuntos
Gammaretrovirus/crescimento & desenvolvimento , Mamíferos , Polimorfismo Genético , Receptores Acoplados a Proteínas-G/genética , Receptores Virais/genética , Tropismo Viral , Substituição de Aminoácidos , Animais , Glicosilação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Virais/metabolismo
5.
Arch Virol ; 164(3): 757-765, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30656465

RESUMO

Koala retrovirus (KoRV) is a gammaretrovirus that is becoming endogenous in koalas. Here, we explored the dynamics of KoRV infection in captive koalas in Japan. We isolated peripheral blood mononuclear cells (PBMCs) from 11 koalas, from which we extracted the KoRV genome. We found the prevalence of KoRV provirus in the koalas to be 100%, and the copy number of KoRV proviral DNA in genomic DNA isolated from PBMCs was variable. The KoRV envelope genes from 11 koalas were sequenced and all were found to be KoRV type A. Nucleotide substitution analysis revealed differences in the KoRV env gene sequences of parents and their offspring. Although no viral KoRV RNA was detected in plasma of healthy koalas, a high copy number was found in plasma of a diseased koala (#6). Hematological analysis showed a high white blood cell (WBC) count in the blood of koala #6. Notably, when retested ~ 5 months later, koala #6 was found to be negative for KoRV in plasma, and the WBC count was within the normal range. Therefore, KoRV in the plasma could be a possible indicator of koala health. We also investigated KoRV growth in concanavalin-A-stimulated koala PBMCs by measuring the KoRV provirus copy number in gDNA and the KoRV RNA copy number in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-culture. We also observed that KoRV isolates were able to infect HEK293T cells. These findings could enhance our understanding of the dynamics of KoRV and its pathogenesis in koalas.


Assuntos
Gammaretrovirus/genética , Gammaretrovirus/isolamento & purificação , Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Animais , Feminino , Gammaretrovirus/classificação , Células HEK293 , Humanos , Japão , Leucócitos Mononucleares/virologia , Masculino , RNA Viral/genética , Infecções por Retroviridae/virologia
6.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541852

RESUMO

Approximately 10% of the mouse genome is composed of endogenous retroviruses belonging to different families. In contrast to the situation in the human genome, several of these families correspond to recent, still-infectious elements capable of encoding complete viral particles. The mouse GLN endogenous retrovirus is one of these active families. We previously identified one fully functional provirus from the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identified receptors for mouse ecotropic retroviral envelope proteins. In this study, we have identified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope is due to the presence or absence of an N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains of the receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses a member of the solute carrier superfamily as a receptor.IMPORTANCE Endogenous retroviruses are genomic traces of past infections present in all vertebrates. Most of these elements degenerate over time and become nonfunctional, but the mouse genome still contains several families with full infection abilities. The GLN retrovirus is one of them, and its members encode particles that are able to infect only mouse cells. Here, we identified the cellular protein used as a receptor by GLN for cell entry. It is SLC19A1, the reduced folate carrier. We show that GLN infection is limited to mouse cells due to both a mutation in the mouse gene preventing the glycosylation of SLC19A1 and also other residues conserved within the rat but not in the hamster and human proteins. Like all other gammaretroviruses whose receptors have been identified, GLN uses a member of the solute carrier superfamily for cell entry, highlighting the role of these proteins for retroviral infection in mammals.


Assuntos
Gammaretrovirus/metabolismo , Produtos do Gene env/genética , Receptores Virais/genética , Proteína Carregadora de Folato Reduzido/genética , Proteínas do Envelope Viral/genética , Ligação Viral , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Gammaretrovirus/genética , Genoma/genética , Glicosilação , Células HEK293 , Especificidade de Hospedeiro , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Proteína Carregadora de Folato Reduzido/metabolismo , Infecções por Retroviridae/virologia
7.
Viruses ; 10(8)2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096897

RESUMO

Naturally-occurring lymphomagenesis is induced by mouse leukemia viruses (MLVs) carried as endogenous retroviruses (ERVs). Replicating the ecotropic MLVs recombines with polytropic (P-ERVs) and xenotropic ERVs (X-ERVs) to generate pathogenic viruses with an altered host range. While most recovered nonecotropic recombinants have a polytropic host range, the X-MLVs are also present in the pre-leukemic tissues. We analyzed two such isolates from the AKR mice to identify their ERV progenitors and to look for evidence of recombination. AKR40 resembles the active X-ERV Bxv1, while AKR6 has a Bxv1-like backbone with substitutions that alter the long terminal repeat (LTR) enhancer and the envelope (env). AKR6 has a modified xenotropic host range, and its Env residue changes all lie outside of the domain that governs the receptor choice. The AKR6 segment spanning the two substitutions, but not the entire AKR6 env-LTR, exists as an ERV, termed Xmv67, in AKR, but not in the C57BL/6 mice. This suggests that AKR6 is the product of one, not two, recombination events. Xmv67 originated in the Asian mice. These data indicate that the recombinant X-MLVs that can be generated during lymphomagenesis, describe a novel X-ERV subtype found in the AKR genome, but not in the C57BL/6 reference genome, and identify residues in the envelope C-terminus that may influence the host range.


Assuntos
Retrovirus Endógenos/genética , Evolução Molecular , Gammaretrovirus/genética , Vírus da Leucemia Murina/genética , Linfoma/virologia , Recombinação Genética , Animais , Gammaretrovirus/isolamento & purificação , Genoma Viral , Especificidade de Hospedeiro , Vírus da Leucemia Murina/isolamento & purificação , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Receptores Virais/genética , Sequências Repetidas Terminais
8.
Front Immunol ; 9: 492, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29616024

RESUMO

There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh) located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV)-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh) cells using antiviral chimeric antigen receptor (CAR) T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure) of HIV and SIV infections.


Assuntos
Linfócitos B/imunologia , Receptores de Antígenos Quiméricos , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/imunologia , Transdução Genética , Replicação Viral/imunologia , Animais , Linfócitos B/patologia , Quimiocina CXCL13/genética , Quimiocina CXCL13/imunologia , Gammaretrovirus , HIV-1/genética , HIV-1/imunologia , Macaca mulatta , Receptores CXCR5/genética , Receptores CXCR5/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Linfócitos T/patologia , Replicação Viral/genética
9.
J Vet Sci ; 19(3): 384-392, 2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-29366300

RESUMO

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.


Assuntos
Gammaretrovirus/fisiologia , Genes MHC Classe I/genética , Antígenos de Histocompatibilidade Classe I/genética , Infecções por Retroviridae/veterinária , Doenças dos Suínos/imunologia , Infecções Tumorais por Vírus/veterinária , Viremia/veterinária , Animais , Animais Geneticamente Modificados , Retrovirus Endógenos/fisiologia , Gammaretrovirus/genética , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Suínos , Doenças dos Suínos/virologia , Transplante Heterólogo , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Viremia/virologia
10.
Microbiol Mol Biol Rev ; 82(1)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29237726

RESUMO

Viruses of the subfamily Orthoretrovirinae are defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and "fossil" endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions.


Assuntos
Retrovirus Endógenos/classificação , Evolução Molecular , Gammaretrovirus/classificação , Infecções por Retroviridae/transmissão , Infecções Tumorais por Vírus/transmissão , Zoonoses/transmissão , Animais , Reservatórios de Doenças , Retrovirus Endógenos/genética , Gammaretrovirus/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Phascolarctidae/virologia , Filogenia , Filogeografia , Ratos , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/virologia , Zoonoses/virologia
11.
J Virol ; 92(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237837

RESUMO

The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV: KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes require population level studies of their prevalence and diversity, especially when coinfected in the same population, and investigations of their modes of transmission in the wild. Toward this end, we studied a wild Queensland koala population of 290 animals over a 5-year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope protein sequence. In contrast, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B-positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B-positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data. IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B-infected/coinfected animals and the linkage of this infection to chlamydial disease, neoplasia, viral evolution, and spread.


Assuntos
Infecções por Chlamydia/epidemiologia , Gammaretrovirus/classificação , Produtos do Gene env/genética , Transmissão Vertical de Doença Infecciosa , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/transmissão , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/veterinária , Sequência de Aminoácidos , Animais , Austrália/epidemiologia , Evolução Molecular , Feminino , Gammaretrovirus/genética , Masculino , Neoplasias/veterinária , Neoplasias/virologia , Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/transmissão , Infecções Tumorais por Vírus/virologia
12.
Sci Rep ; 7(1): 15838, 2017 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-29158564

RESUMO

The koala retrovirus (KoRV) is implicated in several diseases affecting the koala (Phascolarctos cinereus). KoRV provirus can be present in the genome of koalas as an endogenous retrovirus (present in all cells via germline integration) or as exogenous retrovirus responsible for somatic integrations of proviral KoRV (present in a limited number of cells). This ongoing invasion of the koala germline by KoRV provides a powerful opportunity to assess the viral strategies used by KoRV in an individual. Analysis of a high-quality genome sequence of a single koala revealed 133 KoRV integration sites. Most integrations contain full-length, endogenous provirus; KoRV-A subtype. The second most frequent integrations contain an endogenous recombinant element (recKoRV) in which most of the KoRV protein-coding region has been replaced with an ancient, endogenous retroelement. A third set of integrations, with very low sequence coverage, may represent somatic cell integrations of KoRV-A, KoRV-B and two recently designated additional subgroups, KoRV-D and KoRV-E. KoRV-D and KoRV-E are missing several genes required for viral processing, suggesting they have been transmitted as defective viruses. Our results represent the first comprehensive analyses of KoRV integration and variation in a single animal and provide further insights into the process of retroviral-host species interactions.


Assuntos
Evolução Molecular , Phascolarctidae/genética , Infecções por Retroviridae/genética , Retroviridae/genética , Animais , Gammaretrovirus , Células Germinativas , Phascolarctidae/virologia , Retroviridae/patogenicidade , Infecções por Retroviridae/virologia , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
13.
Cell Rep ; 21(1): 17-26, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978471

RESUMO

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.


Assuntos
Ligante 4-1BB/genética , Antígenos de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Mutantes Quiméricas/genética , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Ligante 4-1BB/imunologia , Animais , Antígenos de Neoplasias/imunologia , Morte Celular , Sobrevivência Celular , Gammaretrovirus/genética , Gammaretrovirus/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas Mutantes Quiméricas/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Transplante de Neoplasias , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/patologia , Linfócitos T/transplante , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Receptor fas/genética , Receptor fas/imunologia
14.
Hematol Oncol Clin North Am ; 31(5): 721-735, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28895843

RESUMO

Gene therapy using hematopoietic stem cells (HSC) has developed over the past 3 decades, with progressive improvements in the efficacy and safety. Autologous transplantation of HSC modified with murine gammaretroviral vectors first showed clinical benefits for patients with several primary immune deficiencies, but some of these patients suffered complications from vector-related genotoxicity. Lentiviral vectors have been used recently for gene addition to HSC and have yielded clinical benefits for primary immune deficiencies, metabolic diseases, and hemoglobinopathies, without vector-related complications. Gene editing using site-specific endonucleases is emerging as a promising technology for gene therapy and is moving into clinical trials.


Assuntos
Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Ensaios Clínicos como Assunto , Gammaretrovirus/genética , Edição de Genes , Terapia Genética/efeitos adversos , Terapia Genética/história , Terapia Genética/métodos , Terapia Genética/tendências , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , História do Século XX , História do Século XXI , Recombinação Homóloga , Humanos , Lentivirus/genética
15.
Hematol Oncol Clin North Am ; 31(5): 737-752, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28895844

RESUMO

Gene therapy using autologous or allogeneic cells offers promising possibilities to treat inherited and acquired diseases, ideally leading to a long-lasting therapeutic correction. This article summarizes efforts that use integrating vectors derived from retroviruses and transposons, and briefly explains integrating vector biology and integration site analysis and recent successful application of this technology in clinical trials. Moreover, outlined is how these vectors can be used for cancer gene discovery and clonal tracking of benign and malignant hematopoiesis to gain insights into the dynamics of hematopoiesis.


Assuntos
Rastreamento de Células , Evolução Clonal , Engenharia Genética , Terapia Genética , Vetores Genéticos , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Animais , Ensaios Clínicos como Assunto , Elementos de DNA Transponíveis , Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Lentivirus/genética , Mutagênese Insercional , Transgenes , Integração Viral
16.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28768854

RESUMO

Recombination events induce significant genetic changes, and this process can result in virus genetic diversity or in the generation of novel pathogenicity. We discovered a new recombinant feline leukemia virus (FeLV) gag gene harboring an unrelated insertion, termed the X region, which was derived from Felis catus endogenous gammaretrovirus 4 (FcERV-gamma4). The identified FcERV-gamma4 proviruses have lost their coding capabilities, but some can express their viral RNA in feline tissues. Although the X-region-carrying recombinant FeLVs appeared to be replication-defective viruses, they were detected in 6.4% of tested FeLV-infected cats. All isolated recombinant FeLV clones commonly incorporated a middle part of the FcERV-gamma4 5'-leader region as an X region. Surprisingly, a sequence corresponding to the portion contained in all X regions is also present in at least 13 endogenous retroviruses (ERVs) observed in the cat, human, primate, and pig genomes. We termed this shared genetic feature the commonly shared (CS) sequence. Despite our phylogenetic analysis indicating that all CS-sequence-carrying ERVs are classified as gammaretroviruses, no obvious closeness was revealed among these ERVs. However, the Shannon entropy in the CS sequence was lower than that in other parts of the provirus genome. Notably, the CS sequence of human endogenous retrovirus T had 73.8% similarity with that of FcERV-gamma4, and specific signals were detected in the human genome by Southern blot analysis using a probe for the FcERV-gamma4 CS sequence. Our results provide an interesting evolutionary history for CS-sequence circulation among several distinct ancestral viruses and a novel recombined virus over a prolonged period.IMPORTANCE Recombination among ERVs or modern viral genomes causes a rapid evolution of retroviruses, and this phenomenon can result in the serious situation of viral disease reemergence. We identified a novel recombinant FeLV gag gene that contains an unrelated sequence, termed the X region. This region originated from the 5' leader of FcERV-gamma4, a replication-incompetent feline ERV. Surprisingly, a sequence corresponding to the X region is also present in the 5' portion of other ERVs, including human endogenous retroviruses. Scattered copies of the ERVs carrying the unique genetic feature, here named the commonly shared (CS) sequence, were found in each host genome, suggesting that ancestral viruses may have captured and maintained the CS sequence. More recently, a novel recombinant FeLV hijacked the CS sequence from inactivated FcERV-gamma4 as the X region. Therefore, tracing the CS sequences can provide unique models for not only the modern reservoir of new recombinant viruses but also the genetic features shared among ancient retroviruses.


Assuntos
Regiões 5' não Traduzidas/genética , Retrovirus Endógenos/genética , Genes gag , Genoma Viral , Vírus da Leucemia Felina/genética , Recombinação Genética , Animais , Gatos/virologia , Evolução Molecular , Gammaretrovirus/genética , Humanos/virologia , Leucemia Felina/virologia , Mamíferos/genética , Mamíferos/virologia , Filogenia , Provírus/genética , Provírus/fisiologia , RNA Viral/genética , Suínos/virologia
17.
Virology ; 507: 140-150, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28437635

RESUMO

Porcine endogenous retrovirus-A (PERV-A), a gammaretrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation.


Assuntos
Cisteína/metabolismo , Retrovirus Endógenos/patogenicidade , Gammaretrovirus/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Virais/química , Receptores Virais/metabolismo , Infecções por Retroviridae/veterinária , Doenças dos Suínos/metabolismo , Animais , Cisteína/química , Cisteína/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Gammaretrovirus/classificação , Gammaretrovirus/genética , Glicosilação , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Virais/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Virulência
18.
Elife ; 62017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28397686

RESUMO

Endogenous retroviral sequences provide a molecular fossil record of ancient infections whose analysis might illuminate mechanisms of viral extinction. A close relative of gammaretroviruses, HERV-T, circulated in primates for ~25 million years (MY) before apparent extinction within the past ~8 MY. Construction of a near-complete catalog of HERV-T fossils in primate genomes allowed us to estimate a ~32 MY old ancestral sequence and reconstruct a functional envelope protein (ancHTenv) that could support infection of a pseudotyped modern gammaretrovirus. Using ancHTenv, we identify monocarboxylate transporter-1 (MCT-1) as a receptor used by HERV-T for attachment and infection. A single HERV-T provirus in hominid genomes includes an env gene (hsaHTenv) that has been uniquely preserved. This apparently exapted HERV-T env could not support virion infection but could block ancHTenv mediated infection, by causing MCT-1 depletion from cell surfaces. Thus, hsaHTenv may have contributed to HERV-T extinction, and could also potentially regulate cellular metabolism.


Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Hominidae/genética , Animais , Gammaretrovirus/genética , Gammaretrovirus/crescimento & desenvolvimento , Hominidae/virologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Receptores Virais/metabolismo , Simportadores/metabolismo , Ligação Viral , Internalização do Vírus
20.
Mol Ther ; 25(5): 1132-1141, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28366768

RESUMO

Prior to the first successful bone marrow transplant in 1968, patients born with severe combined immunodeficiency (SCID) invariably died. Today, with a widening availability of newborn screening, major improvements in the application of allogeneic procedures, and the emergence of successful hematopoietic stem and progenitor cell (HSC/P) gene therapy, the majority of these children can be identified and cured. Here, we trace key steps in the development of clinical gene therapy for SCID and other primary immunodeficiencies (PIDs), and review the prospects for adoption of new targets and technologies.


Assuntos
Adenosina Desaminase/genética , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Síndromes de Imunodeficiência/terapia , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/deficiência , Adenosina Desaminase/imunologia , Ensaios Clínicos como Assunto , Gammaretrovirus/genética , Gammaretrovirus/imunologia , Expressão Gênica , Terapia Genética/história , Vetores Genéticos/química , Vetores Genéticos/imunologia , História do Século XX , História do Século XXI , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Lentivirus/genética , Lentivirus/imunologia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Transplante Homólogo
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