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1.
J Viral Hepat ; 18(6): 377-83, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21143343

RESUMO

Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of end-stage liver disease, including cirrhosis, liver failure and primary liver cancer. There are now seven antiviral agents approved by the United States Food and Drug Administration (FDA) for the management of chronic HBV infection. Despite the fact that there are between 1.4 and 2 million chronic HBV infections in the United States, fewer than 50,000 people per year receive prescriptions for HBV antiviral medications. This report discusses possible explanations for the disparity between the number of people who are chronically infected and the number of people who receive treatment. Explanations for this incongruence include the potentially large number of infected persons who are unscreened and thus remain undiagnosed, and lack of access, including insurance, education and referral to appropriate medical care, particularly for disproportionately infected populations.


Assuntos
Antivirais/uso terapêutico , Disparidades em Assistência à Saúde , Hepatite B Crônica/tratamento farmacológico , Doença Hepática Terminal/diagnóstico , Doença Hepática Terminal/tratamento farmacológico , Vírus da Hepatite B , Hepatite B Crônica/diagnóstico , Humanos , Estados Unidos , Vacinação
2.
J Viral Hepat ; 15(1): 42-51, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18088244

RESUMO

This study was conducted to understand the symptomatology, attitudes, and behaviours of chronic hepatitis B (CHB) patients in the USA. CHB patients enrolled in this study were recruited through multiple methods, including newspaper advertisements. Interviews were conducted in multiple languages, and all participants had a history of CHB infection for at least 6 months. Patients with documented human immunodeficiency virus or hepatitis C virus coinfection were excluded from data analyses, resulting in a total study population of 258 respondents who completed interviews between April and June 2004. The majority of monoinfected patients were male (57%) and non-Asian (92%, including 52% Caucasian, 32% African American and others). Length of diagnosis was 5.8 years for all participants (9.1-year Asian and 5.1-year non-Asian). Ninety-five per cent of CHB patients reported symptoms of differing severity in the 12 months prior to the survey. The most common symptoms included fatigue/loss of energy (90%) and loss of appetite (79%). Non-Asian patients described greater symptomatology, and were more likely than Asians to consider CHB an overriding concern in their daily activities. Patients were treated either currently or previously with interferon (IFN) described greater symptomatology than those treated without IFN. Survey results indicate that CHB patients may have greater symptomatology than recognized. Disease perceptions and treatment attitudes differ between Asian and non-Asian ethnic groups, with the former appearing to be more accepting and less concerned about the disease. Additional research about CHB symptomatology and health attitudes by ethnicity is needed to ensure that individuals with CHB are educated on the potential health risks and the availability of current treatment options.


Assuntos
Atitude Frente a Saúde , Hepatite B Crônica/fisiopatologia , Hepatite B Crônica/psicologia , Adulto , Etnicidade , Feminino , Hepatite B Crônica/etnologia , Hepatite B Crônica/terapia , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Índice de Gravidade de Doença , Estados Unidos
3.
DNA Cell Biol ; 20(10): 647-56, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11749723

RESUMO

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.


Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Glucosidases/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Genes Virais , Glicosilação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Tamanho da Partícula , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Transfecção , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genética
4.
Hepatology ; 33(6): 1488-95, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11391538

RESUMO

Previously we have shown that the imino sugar inhibitor of N-linked glycan processing, N-nonyl-deoxynojirimycin (N-nonyl-DNJ), had antiviral activity in the woodchuck model of chronic hepatitis B virus (HBV) infection. In studying the mechanism of action of this compound, it was discovered that imino sugars could inhibit HBV secretion without inhibiting N-linked glycoprocessing. Although N-nonyl-DNJ is an inhibitor of the endoplasmic reticulum (ER) glucosidase, here it is shown that N-nonyl-DNJ retained antiviral activity at concentrations that had no significant impact on ER glucosidase function. Taken together, these results suggested that N-nonyl-DNJ possessed an antiviral activity attributable to a function other than an impact on glycoprocessing. This hypothesis was confirmed by experiments showing that N-nonyl-deoxygalactojirimycin (N-nonyl-DGJ), an alkyl derivative of galactose with no impact on glycoprocessing, retains anti-HBV activity. The data suggest that N-nonyl-DGJ exerts its antiviral action at a point before viral envelopment and may prevent the proper encapsidation of the HBV pregenomic RNA.


Assuntos
1-Desoxinojirimicina/farmacologia , Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Viral/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Hepatite B/genética , 1-Desoxinojirimicina/análogos & derivados , Animais , Bovinos , Linhagem Celular , DNA Viral/antagonistas & inibidores , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/metabolismo , Inibidores de Glicosídeo Hidrolases , Membranas Intracelulares/metabolismo , Polissacarídeos/metabolismo
5.
Antivir Chem Chemother ; 12(6): 317-25, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12018676

RESUMO

Glucosidases in the endoplasmic reticulum (ER) mediate the first step in processing N-linked oligosaccharides. Recent evidence suggests that morphogenesis and secretion of members of the hepatitis B and flavivirus families are more dependent on these enzymes than are most host glycoproteins. Thus, it is possible that glucosidase inhibitors can be designed that are safe and selective for the treatment of hepatitis B and possibly C (since hepatitis C virus is a member of the flavivirus family), making them broad spectrum with respect to hepatitis viruses. Numerous pharmacological and genetic dissections support the notion that glucosidase inhibition can have an antiviral effect, and imino sugars that competitively inhibit ER glucosidases have been proposed as anti-hepatitis drug candidates. We call this family of compounds 'glucovirs'. Recently, however, alkylated imino sugars that retain substantial antiviral activity but lack glucosidase inhibitory activity have been described. These compounds are called 'alkovirs' and their mechanism of action is unknown. This review considers the rationale of the glucovir and alkovir approach to the treatment of hepatitis B and C.


Assuntos
Antivirais/química , Antivirais/farmacologia , Carboidratos/química , Carboidratos/farmacologia , Glucosidases/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucosidases/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Modelos Biológicos , Proteínas Virais/metabolismo
6.
J Neurovirol ; 6(4): 341-9, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10951558

RESUMO

This study reports that quiescent herpes simplex virus (HSV) type 1 genomes, persisting in long-term infected nerve growth factor (NGF) differentiated PC12 cells, were not stimulated by superinfection with a HSV-1. We have previously shown that HSV-1 can establish long term, quiescent infections in NGF differentiated PC12 cells. To determine if virion associated factors or virus induced gene products could trans-activate the quiescent viral genomes, long term infected PC12 cell cultures were superinfected at a high moi (moi of 20) with a recombinant HSV 17alpha47/lacZ that contains the lacZ gene within the alpha 47 locus. Progeny virus and gene expression from the resident 'quiescent' viral genomes were not detected following superinfection with recombinant 17alpha47/lacZ. The failure to stimulate the quiescent genome appears to be related to the inability of the super infecting virus to induce any gene expression from its own genome following entry into the long term NGF treated PC12 cells. Interestingly, both primary and superinfecting viruses could be stimulated from the quiescently infected cultures following cocultivation with inducer cells. These data suggest that (i) HSV genomes in quiescently infected PC12 cells are unable to be stimulated by incoming virion associated factors and (ii) NGF differentiated PC12 cells maintained in tissue culture for longer than 3 weeks became completely refractory to viral gene expression. The possibilities that these results are reflective of populations of neural cells, in vivo in mouse central nervous system, which are completely refractory to virus gene expression, yet accommodating to the maintenance of viral genomes and thus favor 'latency', are discussed.


Assuntos
Herpesvirus Humano 1/fisiologia , Fator de Crescimento Neural/farmacologia , Células PC12/virologia , Latência Viral , Animais , Técnicas de Cocultura , DNA Complementar/análise , DNA Viral/análise , DNA Viral/genética , Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Óperon Lac , Células PC12/efeitos dos fármacos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio de Placa Viral , Ativação Viral
7.
Virology ; 265(2): 296-307, 1999 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-10600601

RESUMO

Herpes Simplex virus expresses latency-associated transcripts (LATs) the function of which remains obscure despite increasing knowledge of their structure and expression. Upstream of the LAT coding region is a region of the genome that is poorly characterized although it lies in an area that is responsible for modulation of reactivation efficiency in two different animal models. Transcript mapping with strains 17, McKrae, KOS, and F has revealed strain differences in this region of the viral genome. Strain 17 and McKrae expressed a novel polyadenylated 0.7-kb transcript that is absent from KOS and F. This transcript is expressed in the LAT direction and has the kinetics of a true late gene during the lytic cycle of infection. A deletion mutant, 17DeltaBsa, which does not express the 0.7-kb RNA, is less virulent than the parental strain 17. A rescuant with F sequence (17DeltaBsa/RF) shows virulence similar to F, whereas a rescuant with strain 17 sequence (17DeltaBsa/R17) is similar to strain 17. Virulence is altered by deletion or substitution in the region encoding the 0.7-kb transcript (BsaI-BsaI); however, reactivation in the mouse explant cocultivation assay or the adrenergically induced rabbit reactivation model remained unchanged. The importance of this region for virulence is discussed.


Assuntos
Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Regiões Promotoras Genéticas , RNA Viral/genética , Animais , Chlorocebus aethiops , Mapeamento Cromossômico , Expressão Gênica , Genoma Viral , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/isolamento & purificação , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Poli A/genética , Coelhos , Transcrição Gênica , Células Tumorais Cultivadas , Células Vero , Virulência/genética , Ativação Viral , Latência Viral/genética
8.
Proc Natl Acad Sci U S A ; 96(21): 11878-82, 1999 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-10518544

RESUMO

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells alpha-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.


Assuntos
Antivirais/metabolismo , Vírus da Diarreia Viral Bovina/metabolismo , Desenho de Fármacos , Hepacivirus/metabolismo , Pestivirus/metabolismo , 1-Desoxinojirimicina/farmacologia , Animais , Bovinos , Sobrevivência Celular , Retículo Endoplasmático/virologia , Inibidores de Glicosídeo Hidrolases , Hepacivirus/efeitos dos fármacos , Humanos , Polissacarídeos/metabolismo , Distribuição Tecidual
9.
J Virol ; 73(5): 4171-80, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10196313

RESUMO

A two-cell system for the stimulation of herpes simplex virus type 1 (HSV-1) from an in vitro model of long-term (quiescent) infection is described. Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor were infected with HSV-1 strain 17. Little, if any, cytotoxicity was observed, and a quiescent infection was established. The long-term infection was characterized by the absence of all detectable virus in the culture medium and little, if any, detectable early or late viral-gene expression as determined by reverse transcriptase PCR analysis. The presence of HSV-1 DNA was determined by PCR analysis. This showed that approximately 180 viral genomes were present in limiting dilutions where as few as 16 cells were examined. The viral DNA was infectious, since cocultivation with human corneal fibroblasts (HCF) or human corneal epithelial cells (HCE) resulted in recovery of virus from most, if not all, clusters of PC12 cells. Following cocultivation, viral antigens appeared first on PC12 cells and then on neighboring inducing cells, as determined by immunofluorescent staining, demonstrating that de novo viral protein synthesis first occurred in the long-term-infected PC12 cells. Interestingly, the ability to induce HSV varied among the cell lines tested. For example, monkey kidney CV-1 cells and human hepatoblastoma HepG2 cells, but not mouse neuroblastoma cells or undifferentiated PC12 cells, mediated stimulation. This work thus shows that (i) quiescent HSV infections can be maintained in PC12 cells in vitro, (ii) HSV can be induced from cells which do not accumulate significant levels of latency-associated transcripts, and (iii) the activation of HSV gene expression can be induced via neighboring cells. The ability of adjacent cells to stimulate HSV gene expression in neuron-like cells represents a novel area of study. The mechanism(s) whereby HCF, HCE, and HepG2 and CV-1 cells communicate with PC12 cells and stimulate viral replication, as well as how this system compares with other in vitro models of long-term infection, is discussed.


Assuntos
Córnea/citologia , Fibroblastos/fisiologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Latência Viral , Animais , Antígenos Virais , Linhagem Celular , Chlorocebus aethiops , Técnicas de Cocultura , Expressão Gênica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Camundongos , Fatores de Crescimento Neural/farmacologia , Células PC12 , Ratos , Fatores de Tempo , Células Tumorais Cultivadas , Ativação Viral
10.
J Virol ; 73(1): 767-71, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9847385

RESUMO

The herpes simplex virus type 1 (HSV-1) LAT gene is the only viral gene abundantly transcribed during latency. LAT null mutants created with strains McKrae and 17syn+ are impaired for both in vivo spontaneous and in vivo-induced reactivation. Thus, LAT is essential for efficient in vivo-induced and spontaneous reactivation. Different investigators have studied two LAT mutants containing a StyI-StyI region deletion corresponding to LAT nucleotides 76 to 447. One mutant, dLAT371 (parent strain, McKrae), had parental high frequencies of spontaneous reactivation. In vivo-induced reactivation was not examined. The other mutant, 17DeltaSty (parent strain, 17syn+), had parental frequencies of in vitro reactivation following cocultivation of explanted ganglia but reduced frequencies of in vivo-induced reactivation. Spontaneous reactivation frequency was not reported for 17DeltaSty. These combined results suggested the possibility that in vivo spontaneous reactivation and in vivo-induced reactivation may map to different regions within the LAT domain. We now report that dLAT371 has in vivo-induced reactivation frequencies of the parent and that 17DeltaSty has reduced frequencies of in vivo spontaneous reactivation. Thus, dLAT371 demonstrated the parental phenotype for both in vivo spontaneous and -induced reactivation while the apparently identical 17DeltaSty was impaired for both in vivo spontaneous and -induced reactivation. These results suggest that one or more differences between the genetic backgrounds of McKrae and 17syn+ result in different in vivo reactivation phenotypes of otherwise identical deletion mutations and that McKrae may have compensating sequences sufficient to overcome the loss of the StyI-StyI region of the LAT transcript.


Assuntos
Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Ativação Viral , Latência Viral/genética , Animais , Pareamento de Bases , Genes Virais , Mutação , Fenótipo , Coelhos
11.
FEBS Lett ; 430(1-2): 17-22, 1998 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-9678587

RESUMO

N-Linked oligosaccharides play many roles in the fate and functions of glycoproteins. One function is to assist in the folding of proteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases and this causes some proteins to be misfolded and retained within the endoplasmic reticulum. In human immunodeficiency virus (HIV) and hepatitis B virus (HBV) the misfolding of key viral envelope glycoproteins interferes with the viral life cycle. It has been demonstrated in an animal model of chronic HBV that glucosidase inhibitors can alter glycosylation and have anti-viral activity. As the mechanism of action of alpha-glucosidase inhibitors is the induction of misfolded or otherwise defective viral glycoproteins, such inhibitors may be useful therapeutics for many viruses, especially those which bud from the endoplasmic reticulum (where protein folding takes place). For example bovine viral diarrhea virus, a pestivirus akin to hepatitis C virus, is also extremely sensitive to glucosidase inhibition.


Assuntos
Antivirais/farmacologia , Inibidores de Glicosídeo Hidrolases , Animais , Bovinos , Retículo Endoplasmático/enzimologia , Humanos , Chaperonas Moleculares/metabolismo , Proteínas/metabolismo
12.
Clin Chim Acta ; 273(1): 43-58, 1998 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-9620469

RESUMO

The solubility of ammonia, alphaNH3 (mM/mmHg), was determined at 37 degrees C and low ammonia partial pressure (0.02-1 mmHg) in pure water (n =24) as 46.70+/-0.40; aqueous isotonic salt solutions (n = 7) as 46.8+/-0.81; and human plasma (n = 5) as 42.0+/-0.66. The last figure increases to 45.3+/-0.63 if expressed in molal units (mmol/kg plasma water x mmHg) instead of molarity with respect to the water content of the plasma (mean from four healthy and fasting donors: 0.908+0.005 kg H2O/kg plasma; mean density at 37 degrees C: 1.020+/-0.002 kg/l). In pure water, the solubility value is the mean of three different methods: (a) extrapolation of the salting-out effect of ammonia in aqueous NaOH to zero concentration; (b) slope of Henry-Dalton's law and (c) directly measured in pure water and 0.001 M aqueous NaOH. Based on the Henderson-Hasselbalch equation for the system NH4/NH3 in isotonic salt solutions and human plasma, both constants, apparent pK and solubility, can be derived from total ammonia concentration and pH at equilibrium with defined ammonia gas phase, if additionally the concentration of NH4 or NH3 is known. This was verified, in the first case, by determining the concentration of NH4+ by the experimental conditions, and in the second, by two measurements of total ammonia concentration at two different pH values. Total ammonia concentration was measured by a specific enzymatic standard test and pH with the glass electrode. The mean apparent pK was 8.968+/-0.013 in isotonic salt solutions (n = 7), and in human plasma (n = 10) it was 9.014+/-0.033.


Assuntos
Amônia/química , Água/química , Amônia/sangue , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Cloreto de Sódio/química , Solubilidade , Soluções
13.
Nat Med ; 4(5): 610-4, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9585237

RESUMO

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.


Assuntos
1-Desoxinojirimicina/análogos & derivados , Antivirais/uso terapêutico , Inibidores de Glicosídeo Hidrolases , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B Crônica/veterinária , Doenças dos Roedores/terapia , 1-Desoxinojirimicina/uso terapêutico , Animais , Transporte Biológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Glucosídeos/sangue , Glicosilação , Hepatite B Crônica/terapia , Manosídeos/sangue , Marmota , Oligossacarídeos/sangue , Dobramento de Proteína , Replicação Viral/efeitos dos fármacos
14.
Virology ; 243(1): 140-9, 1998 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-9527923

RESUMO

We previously have shown that two latency-associated transcripts (LATs) of herpes simplex type 1 (HSV-1) are probably lariats, produced during splicing. By RNaseH digestion analysis, we now show that the major branchpoint of the 2.0-kb LAT was within 46 nt 5' of the splice acceptor site. A more detailed mapping by primer extension revealed the branchpoint as an adenosine 29 nt 5' of the splice acceptor site. Introduction of two branchpoint sequences with good matches to the consensus at position -25 had no effect on the splicing efficiency but reduced the accumulation of the 2.0-kb LATs at least 90-fold. The second focus of our studies was the 1.5-kb LAT. It was not detected by Northern analyses in either productively infected or transfected cultured cells or even in cells of neuronal origin. However, it was detected in the trigeminal ganglia of mice experimentally infected with HSV-1 after 10 days. Moreover, its abundance relative to that of the 2.0-kb species increased 4-fold from 10 to 30 days after infection, consistent with an interpretation that the 1.5-kb species, once formed, was more stable than the 2.0-kb species.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Splicing de RNA , RNA Viral/genética , Latência Viral/genética , Animais , Linhagem Celular , Camundongos , Mutação , Transfecção
17.
J Virol ; 71(9): 6555-9, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9261376

RESUMO

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.


Assuntos
Herpesvirus Humano 1/genética , Ceratite Herpética/virologia , Regiões Promotoras Genéticas , Ativação Viral/genética , Agonistas Adrenérgicos/farmacologia , Animais , Composição de Bases , Southern Blotting , Linhagem Celular , Chlorocebus aethiops , Córnea/virologia , DNA Viral/metabolismo , Epinefrina/farmacologia , Genótipo , Humanos , Coelhos , Deleção de Sequência , Gânglio Trigeminal/virologia , Células Vero , Ativação Viral/efeitos dos fármacos , Latência Viral
18.
Proc Natl Acad Sci U S A ; 94(5): 1822-7, 1997 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-9050863

RESUMO

The role of N-linked glycosylation and glycan trimming in the function of glycoproteins remains a central question in biology. Hepatitis B virus specifies three glycoproteins (L, M, and S) that are derived from alternate translation of the same ORF. All three glycoproteins contain a common N-glycosylation site in the S domain while M possesses an additional N-glycosylation site at its amino terminus. In the presence of N-butyl-deoxnojirimycin (an inhibitor of alpha-glucosidase) virions and the M protein are surprisingly retained. Preliminary evidence suggests that the retained M protein is hyperglucosylated and localized to lysosomal vesicles. In contrast, the S and L proteins are secreted, and their glycosylation state is unaffected by the presence of the inhibitor. Site-directed mutagenesis provides evidence that virion secretion requires the glycosylation sequon in the pre-S2 domain of M. This highlights the potential role of the M protein oligosaccharide as a therapeutic target.


Assuntos
Vírus da Hepatite B/metabolismo , Polissacarídeos/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Antígenos de Superfície/análise , Southern Blotting , Carcinoma Hepatocelular , Centrifugação com Gradiente de Concentração , DNA Viral/metabolismo , Inibidores de Glicosídeo Hidrolases , Glicosilação , Humanos , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Transfecção , Células Tumorais Cultivadas , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química
19.
Proc Natl Acad Sci U S A ; 94(6): 2380-5, 1997 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-9122203

RESUMO

The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which alpha-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse-chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein-protein interactions and trafficking are the result of misfolding.


Assuntos
Glicoproteínas/metabolismo , Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Processamento de Proteína Pós-Traducional , Linhagem Celular , DNA Viral/metabolismo , Glucosidases/antagonistas & inibidores , Glucosidases/metabolismo , Glicoproteínas/biossíntese , Glicosilação , Antígenos da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/isolamento & purificação , Humanos , Cinética , Frações Subcelulares/metabolismo , Vírion/fisiologia
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