RESUMO
In cocaine use disorder, relapse can be elicited by drug-associated cues despite long periods of abstinence. The persistence of drug-associated cues in eliciting drug seeking suggests enduring changes in structural and functional plasticity, which may be mediated by basic fibroblast growth factor (bFGF, FGF2). Stimulant drug use increases bFGF expression in reward- and learning-related brain regions, such as the infralimbic medial-prefrontal cortex (IL-mPFC), and we previously found that this increase was reversed by extinction. However, whether bFGF expression is similarly modified in other brain regions is unknown. Therefore, we used the conditioned place preference (CPP) paradigm to assess bFGF expression following cocaine-associated CPP or extinction of that CPP within the mPFC, nucleus accumbens (NAc), hippocampus (Hipp), and basolateral amygdala (BLA). bFGF expression was increased in IL-mPFC and NAc-Core and -Shell following a cocaine-associated CPP, an effect reversed by extinction. Conversely, bFGF expression was increased in BLA following extinction, but no significant changes were observed in PL-mPFC or either dorsal or ventral Hipp. These results demonstrate differential regulation of bFGF following cocaine-associated CPP or extinction of that CPP in discrete brain regions. Changes in bFGF expression may regulate long-lasting drug-induced plasticity that underlies persistent drug-associated memories, and therefore present potential prophylactic targets.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Comportamento Animal/efeitos dos fármacos , Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Extinção Psicológica/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos , Hipocampo , Núcleo Accumbens , Córtex Pré-Frontal , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Complexo Nuclear Basolateral da Amígdala/metabolismo , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Long-EvansRESUMO
UNLABELLED: The human cytomegalovirus (HCMV) major immediate early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), the first 3 exons, and the first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one is essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's fourth intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a chromatin immunoprecipitation (ChIP) assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target. IMPORTANCE: CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than two proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified two Pys in intron 4 and found that the first Py is important and the second is required for the splicing and expression of IE2. We further investigated the mechanisms of gene switching from IE1 to IE2 and found that the two Pys are responsible for U2AF65's binding with intron 4. Therefore, the Pys in intron 4 are the cis elements that determine the fate of IE2 splicing. Furthermore, we found that a small RNA that is complementary to intron 4 repressed IE2 expression. Hence, we provide the first piece of evidence for a unique mechanism of MIE gene regulation at the splicing level.
Assuntos
Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Íntrons , Transativadores/metabolismo , Transcrição Gênica , Replicação Viral , Citomegalovirus/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas Imediatamente Precoces/genética , Ligação Proteica , Pirimidinas/metabolismo , Fatores de Processamento de RNA/metabolismo , Transativadores/genéticaRESUMO
BACKGROUND: HIV-infected individuals are at an increased risk of developing neurological abnormalities. HIV induces neurotoxicity by host cellular factors and individual viral proteins. Some of these proteins including viral protein R (Vpr) promote immune activation and neuronal damage. Vpr is known to contribute to cell death of cultured rat hippocampal neurons and suppresses axonal growth. Behavioral studies are limited and suggest hyperactivity in the presence of Vpr. Thus Vpr may play a role in hippocampal loss of function. The purpose of this study is to determine the ability of HIV-1 Vpr production by astrocytes in the hippocampus to cause neurological deficits and memory impairments. METHODS: We tested the performance of rats in novel object and novel location tasks after hippocampal infusion with astrocytes expressing HIV-1 Vpr. Synaptic injury and morphological changes were measured by synaptophysin immunoreactivity and Nissl staining. RESULTS: Vpr-infused rats showed impaired novel location and novel object recognition compared with control rats expressing green fluorescent protein (GFP). This impairment was correlated with a significant decrease in synaptophysin immunoreactivity in the hippocampal CA3 region, suggesting synaptic injury in HIV-1 Vpr-treated animals. In addition, Nissl staining showed morphological changes indicative of neuronal chromatolysis in the Vpr group. The Vpr-induced neuronal damage and synaptic loss suggest that neuronal dysfunction caused the spatial and recognition memory deficits found in the Vpr-infused animals. CONCLUSIONS: In this study, we demonstrate that HIV-1 Vpr produced by astrocytes in the hippocampus impairs hippocampal-dependent learning. The data suggest Vpr is a neurotoxin with the potential to cause learning impairment in HIV-1 infected individuals even under conditions of limited viral replication.
Assuntos
Astrócitos/metabolismo , Hipocampo/patologia , Transtornos da Memória/etiologia , Neurônios/patologia , Sinapses/patologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Aprendizagem em Labirinto , Neurônios/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Percepção Espacial , Sinaptofisina/metabolismo , Transdução Genética , TransfecçãoRESUMO
Human herpesviruses cause different infectious diseases, resulting in world-wide health problems. Sexual transmission is a major route for the spread of both herpes simplex virus-1 (HSV-1) and -2. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa and, thereby, initiates viral replication. Previously, we demonstrated that the amyloid fibrils semenogelin (SEM) and semen-derived enhancer of viral infection (SEVI), and seminal plasma (SP) augment cytomegalovirus infection (Tang et al., J. Virol 2013). Whether SEM or SEVI amyloids or SP could also enhance other herpesvirus infections has not been examined. In this study, we found that the two amyloids as well as SP strongly enhance both HSV-1 and -2 infections in cell culture. Along with SP, SEM and SEVI amyloids enhanced viral entry and increased infection rates by more than 10-fold, as assessed by flow cytometry assay and fluorescence microscopy. Viral replication was increased by about 50- to 100-fold. Moreover, viral growth curve assays showed that SEM and SEVI amyloids, as well as SP, sped up the kinetics of HSV replication such that the virus reached its replicative peak more quickly. The interactions of SEM, SEVI, and SP with HSVs are direct. Furthermore, we discovered that the enhancing effects of SP, SEM, and SEVI can be significantly reduced by heparin, a sulfated polysaccharide with an anionic charge. It is probable that heparin abrogates said enhancing effects by interfering with the interaction of the viral particle and the amyloids, which interaction results in the binding of the viral particles and both SEM and SEVI.
Assuntos
Amiloide/metabolismo , Interações Hospedeiro-Patógeno , Sêmen/metabolismo , Proteínas Secretadas pela Vesícula Seminal/metabolismo , Simplexvirus/efeitos dos fármacos , Simplexvirus/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Citometria de Fluxo , Heparina/metabolismo , Humanos , Microscopia de Fluorescência , Replicação ViralRESUMO
Three crucial hurdles hinder studies on human cytomegalovirus (HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus-host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host (latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate-early (IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate-early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.