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1.
Biologicals ; 71: 1-8, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34039532

RESUMO

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.


Assuntos
Retrovirus Endógenos , Gammaretrovirus , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidade , Gammaretrovirus/genética , Gammaretrovirus/patogenicidade , Ilhotas Pancreáticas/virologia , Suínos , Transplante Heterólogo
2.
Nat Commun ; 12(1): 1316, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637755

RESUMO

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.


Assuntos
Células Germinativas , Neoplasias/genética , Infecções por Retroviridae/genética , Retroviridae/genética , Animais , Retrovirus Endógenos , Evolução Molecular , Gammaretrovirus/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Neoplasias/virologia , Phascolarctidae/genética , Phascolarctidae/virologia , Proteínas Repressoras/genética , Infecções por Retroviridae/virologia , Proteína bcl-X/genética
3.
Viruses ; 13(1)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477490

RESUMO

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Polieletrólitos/química , Retroviridae/fisiologia , Montagem de Vírus , Alpharetrovirus/fisiologia , Animais , Betaretrovirus/fisiologia , Células Cultivadas , Gammaretrovirus/fisiologia , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Polieletrólitos/metabolismo , Retroviridae/ultraestrutura , Vírion
4.
Retrovirology ; 17(1): 34, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33008414

RESUMO

BACKGROUND: Koalas are infected with the koala retrovirus (KoRV) that exists as exogenous or endogenous viruses. KoRV is genetically diverse with co-infection with up to ten envelope subtypes (A-J) possible; KoRV-A is the prototype endogenous form. KoRV-B, first found in a small number of koalas with an increased leukemia prevalence at one US zoo, has been associated with other cancers and increased chlamydial disease. To better understand the molecular epidemiology of KoRV variants and the effect of increased viral loads (VLs) on transmissibility and pathogenicity we developed subtype-specific quantitative PCR (qPCR) assays and tested blood and tissue samples from koalas at US zoos (n = 78), two Australian zoos (n = 27) and wild-caught (n = 21) in Australia. We analyzed PCR results with available clinical, demographic, and pedigree data. RESULTS: All koalas were KoRV-A-infected. A small number of koalas (10.3%) at one US zoo were also infected with non-A subtypes, while a higher non-A subtype prevalence (59.3%) was found in koalas at Australian zoos. Wild koalas from one location were only infected with KoRV-A. We observed a significant association of infection and plasma VLs of non-A subtypes in koalas that died of leukemia/lymphoma and other neoplasias and report cancer diagnoses in KoRV-A-positive animals. Infection and VLs of non-A subtypes was not associated with age or sex. Transmission of non-A subtypes occurred from dam-to-offspring and likely following adult-to-adult contact, but associations with contact type were not evaluated. Brief antiretroviral treatment of one leukemic koala infected with high plasma levels of KoRV-A, -B, and -F did not affect VL or disease progression. CONCLUSIONS: Our results show a significant association of non-A KoRV infection and plasma VLs with leukemia and other cancers. Although we confirm dam-to-offspring transmission of these variants, we also show other routes are possible. Our validated qPCR assays will be useful to further understand KoRV epidemiology and its zoonotic transmission potential for humans exposed to koalas because KoRV can infect human cells.


Assuntos
Gammaretrovirus/genética , Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , Animais Selvagens , Animais de Zoológico , Austrália/epidemiologia , Feminino , Gammaretrovirus/classificação , Gammaretrovirus/isolamento & purificação , Gammaretrovirus/patogenicidade , Variação Genética , Masculino , Epidemiologia Molecular , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Viral/genética , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/transmissão , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/transmissão , Infecções Tumorais por Vírus/virologia , Estados Unidos/epidemiologia , Carga Viral
5.
mBio ; 11(5)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934084

RESUMO

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.


Assuntos
Quirópteros/imunologia , Gammaretrovirus/imunologia , Imunidade Inata/imunologia , Lentivirus de Primatas/imunologia , Spumavirus/imunologia , Células 3T3 , Animais , Aotidae , Gatos , Linhagem Celular , Quirópteros/virologia , Ciclofilina A/metabolismo , Furões , Gammaretrovirus/crescimento & desenvolvimento , Células HEK293 , Humanos , Lentivirus de Primatas/crescimento & desenvolvimento , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Spumavirus/crescimento & desenvolvimento , Proteínas com Motivo Tripartido/metabolismo
6.
Med Microbiol Immunol ; 209(6): 681-691, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32918599

RESUMO

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.


Assuntos
Repetição de Anquirina , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV/isolamento & purificação , Imunoterapia Adotiva , Depleção Linfocítica/métodos , Peptídeos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Relação Dose-Resposta Imunológica , Avaliação Pré-Clínica de Medicamentos , Gammaretrovirus/genética , Vetores Genéticos/genética , Células HEK293 , Infecções por HIV/virologia , Humanos , Ativação Linfocitária , Peptídeos/química , Anticorpos de Cadeia Única/imunologia , Transdução Genética
7.
Sci Rep ; 10(1): 15013, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929174

RESUMO

Chlamydial disease control is increasingly utilised as a management tool to stabilise declining koala populations, and yet we have a limited understanding of the factors that contribute to disease progression. To examine the impact of host and pathogen genetics, we selected two geographically separated south east Queensland koala populations, differentially affected by chlamydial disease, and analysed koala major histocompatibility complex (MHC) genes, circulating strains of Chlamydia pecorum and koala retrovirus (KoRV) subtypes in longitudinally sampled, well-defined clinical groups. We found that koala immunogenetics and chlamydial genotypes differed between the populations. Disease progression was associated with specific MHC alleles, and we identified two putative susceptibility (DCb 03, DBb 04) and protective (DAb 10, UC 01:01) variants. Chlamydial genotypes belonging to both Multi-Locus Sequence Typing sequence type (ST) 69 and ompA genotype F were associated with disease progression, whereas ST 281 was associated with the absence of disease. We also detected different ompA genotypes, but not different STs, when long-term infections were monitored over time. By comparison, KoRV profiles were not significantly associated with disease progression. These findings suggest that chlamydial genotypes vary in pathogenicity and that koala immunogenetics and chlamydial strains are more directly involved in disease progression than KoRV subtypes.


Assuntos
Infecções por Chlamydia/veterinária , Chlamydia/genética , Complexo Principal de Histocompatibilidade/genética , Phascolarctidae/genética , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Técnicas de Tipagem Bacteriana , Chlamydia/classificação , Chlamydia/isolamento & purificação , Infecções por Chlamydia/epidemiologia , Coinfecção , Feminino , Gammaretrovirus/genética , Haplótipos , Interações Hospedeiro-Patógeno/genética , Imunogenética , Complexo Principal de Histocompatibilidade/imunologia , Tipagem de Sequências Multilocus , Phascolarctidae/imunologia , Prevalência , Queensland/epidemiologia , Infecções por Retroviridae/veterinária
8.
Arch Virol ; 165(11): 2409-2417, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32770481

RESUMO

Koala retrovirus (KoRV) is a major threat to koala health and conservation. It also represents a series of challenges across the fields of virology, immunology, and epidemiology that are of great potential interest to any researcher in the field of retroviral diseases. KoRV is a gammaretrovirus that is present in both endogenous and exogenous forms in koala populations, with a still-active endogenization process. KoRV may induce immunosuppression and neoplastic conditions such as lymphoma and leukemia and play a role in chlamydiosis and other diseases in koalas. KoRV transmission modes, pathogenesis, and host immune response still remain unclear, and a clear understanding of these areas is critical for devising effective preventative and therapeutic strategies. Research on KoRV is clearly critical for koala conservation. In this review, we provide an overview of the current understanding and future challenges related to KoRV epidemiology, transmission mode, pathogenesis, and host immune response and discuss prospects for therapeutic and preventive vaccines.


Assuntos
Gammaretrovirus/classificação , Transmissão Vertical de Doenças Infecciosas , Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Sequência de Aminoácidos , Animais , Austrália/epidemiologia , Infecções por Chlamydia/veterinária , Infecções por Chlamydia/virologia , Evolução Molecular , Neoplasias/veterinária , Neoplasias/virologia , Phascolarctidae/imunologia , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/transmissão
9.
J Comp Pathol ; 176: 50-66, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32359636

RESUMO

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.


Assuntos
Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , Austrália , Gammaretrovirus , Austrália do Sul
10.
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
11.
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
12.
Methods Mol Biol ; 2086: 119-130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31707671

RESUMO

Manufacturing chimeric antigen receptor (CAR)-modified T cells requires incorporation of the CAR transgene, for which viral vectors are most often used. Here, we describe the generation of CAR T cells using primary human T cells and a non-self-inactivating gammaretroviral vector encoding a CAR transgene. The gammaretroviral vector is produced by 293T cells transiently transfected with DNA plasmids encoding necessary components of the viral vector. The resulting viral particles efficiently infect activated T cells and integrate the CAR transgene into the genome of dividing cells for stable expression.


Assuntos
Gammaretrovirus/genética , Vetores Genéticos/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Animais , Linhagem Celular , Expressão Gênica , Engenharia Genética , Vetores Genéticos/biossíntese , Humanos , Imunofenotipagem , Imunoterapia Adotiva , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Transdução Genética , Transgenes
13.
Genomics ; 112(1): 886-896, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31175981

RESUMO

Endogenous retroviruses (ERVs) constitute a significant part of vertebrate genomes. They originated from past retroviral infections and some of them retain transcriptional activity. The key mechanism avoiding uncontrolled ERV transcription is DNA methylation-mediated epigenetic silencing. Despite numerous studies describing the involvement of ERV activity in cellular processes, epigenetic regulation of ERVs is still poorly understood. We previously described a cervid endogenous retrovirus (CrERV) in the mule deer genome. This virus exhibits massive insertional polymorphism, suggesting recent activity. Here we employed NGS-based strategy to determine the methylation pattern of CrERV integrations in four mule deer. Besides the vast majority of methylated integrations, we identified a tiny fraction of demethylated proviral copies. These copies represent evolutionary older integrations located near gene promoters. In general, our work is a first attempt to characterize the epigenetic landscape of insertionally polymorphic ERV on a whole-genome scale and offers insight into its interactions with a host.


Assuntos
Cervos/genética , Retrovirus Endógenos , Epigênese Genética , Gammaretrovirus/genética , Animais , Metilação de DNA , Sequências Repetidas Terminais , Integração Viral
14.
J Mol Biol ; 431(24): 4922-4940, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31711961

RESUMO

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the "SOSIP" mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.


Assuntos
Produtos do Gene env/química , Modelos Moleculares , Proteínas da Gravidez/química , Conformação Proteica , Sequência de Aminoácidos , Cristalografia por Raios X , Gammaretrovirus , Produtos do Gene env/metabolismo , Humanos , Proteínas da Gravidez/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas do Envelope Viral/química
15.
Hum Gene Ther ; 30(12): 1477-1493, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31578886

RESUMO

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antígenos CD20/genética , Linfócitos B/virologia , Gammaretrovirus/genética , Vetores Genéticos/biossíntese , Vetores Genéticos/uso terapêutico , Glicoproteínas/genética , Células-Tronco Hematopoéticas/virologia , Humanos , Lentivirus/genética , Vírus da Leucemia do Macaco Gibão/genética , Vírus do Sarampo/genética , Peptídeos/genética , Retroviridae/genética , Linfócitos T/virologia , Transdução Genética , Proteínas do Envelope Viral/genética
16.
Cell ; 179(3): 632-643.e12, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31607510

RESUMO

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.


Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismo
17.
J Virol ; 93(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31534037

RESUMO

Endogenous retroviruses (ERVs) of domestic cats (ERV-DCs) are one of the youngest feline ERV groups in domestic cats (Felis silvestris catus); some members are replication competent (ERV-DC10, ERV-DC18, and ERV-DC14), produce the antiretroviral soluble factor Refrex-1 (ERV-DC7 and ERV-DC16), or can generate recombinant feline leukemia virus (FeLV). Here, we investigated ERV-DC in European wildcats (Felis silvestris silvestris) and detected four loci: ERV-DC6, ERV-DC7, ERV-DC14, and ERV-DC16. ERV-DC14 was detected at a high frequency in European wildcats; however, it was replication defective due to a single G → A nucleotide substitution, resulting in an E148K substitution in the ERV-DC14 envelope (Env). This mutation results in a cleavage-defective Env that is not incorporated into viral particles. Introduction of the same mutation into feline and murine infectious gammaretroviruses resulted in a similar Env dysfunction. Interestingly, the same mutation was found in an FeLV isolate from naturally occurring thymic lymphoma and a mouse ERV, suggesting a common mechanism of virus inactivation. Refrex-1 was present in European wildcats; however, ERV-DC16, but not ERV-DC7, was unfixed in European wildcats. Thus, Refrex-1 has had an antiviral role throughout the evolution of the genus Felis, predating cat exposure to feline retroviruses. ERV-DC sequence diversity was present across wild and domestic cats but was locus dependent. In conclusion, ERVs have evolved species-specific phenotypes through the interplay between ERVs and their hosts. The mechanism of viral inactivation may be similar irrespective of the evolutionary history of retroviruses. The tracking of ancestral retroviruses can shed light on their roles in pathogenesis and host-virus evolution.IMPORTANCE Domestic cats (Felis silvestris catus) were domesticated from wildcats approximately 9,000 years ago via close interaction between humans and cats. During cat evolution, various exogenous retroviruses infected different cat lineages and generated numerous ERVs in the host genome, some of which remain replication competent. Here, we detected several ERV-DC loci in Felis silvestris silvestris Notably, a species-specific single nucleotide polymorphism in the ERV-DC14 env gene, which results in a replication-defective product, is highly prevalent in European wildcats, unlike the replication-competent ERV-DC14 that is commonly present in domestic cats. The presence of the same lethal mutation in the env genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.


Assuntos
Animais Selvagens/virologia , Gatos/virologia , Retrovirus Endógenos/genética , Infecções por Retroviridae/virologia , Animais , Doenças do Gato/imunologia , Doenças do Gato/virologia , Linhagem Celular , Evolução Molecular , Gammaretrovirus/genética , Genes env/genética , Células HEK293 , Humanos , Vírus da Leucemia Felina/genética , Proteínas de Membrana , Camundongos , Mutação , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade da Espécie , Replicação Viral
18.
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
19.
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
20.
J Virol ; 93(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31243137

RESUMO

Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis, or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D, and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ∼10-fold less and KoRV-B expression being ∼100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas than in koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared "vulnerable" by the Australian Government and officially listed as a threatened species. KoRV is clearly contributing to the overall health status of koalas, and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.


Assuntos
Gammaretrovirus/genética , Phascolarctidae/virologia , Retroviridae/genética , Animais , Austrália , Evolução Biológica , Evolução Molecular , Feminino , Regulação Viral da Expressão Gênica/genética , Marsupiais/virologia , Phascolarctidae/metabolismo , Provírus/genética , Retroviridae/metabolismo , Infecções por Retroviridae/virologia
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