RESUMO
The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.
Assuntos
Citomegalovirus/fisiologia , Endossomos/fisiologia , Endossomos/virologia , Montagem de Vírus , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Antígenos CD/análise , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular , Citomegalovirus/crescimento & desenvolvimento , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/genética , Inativação Gênica , Guanilato Quinases , Humanos , Microscopia Imunoeletrônica , Glicoproteínas da Membrana de Plaquetas/análise , Proteínas/genética , Proteínas/metabolismo , Tetraspanina 30 , ATPases Vacuolares Próton-Translocadoras , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genética , Vírion/químicaRESUMO
Human cytomegalovirus can cause a diverse range of diseases in different immunocompromised hosts. The pathogenic mechanisms underlying these diseases have not been fully elucidated, though the maximal viral load during infection is strongly correlated with the disease. However, concentrating on single viral load measures during infection ignores valuable information contained during the entire replication history up to the onset of disease. We use a statistical model that allows all viral load data sampled during infection to be analysed, and have applied it to four immunocompromised groups exhibiting five distinct cytomegalovirus-related diseases. The results show that for all diseases, peaks in viral load contribute less to disease progression than phases of low virus load with equal amount of viral turnover. The model accurately predicted the time of disease onset for fever, gastrointestinal disease and pneumonitis but not for hepatitis and retinitis, implying that other factors may be involved in the pathology of these diseases.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Hospedeiro Imunocomprometido , Modelos Estatísticos , Replicação Viral , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/imunologia , Retinite por Citomegalovirus/imunologia , Retinite por Citomegalovirus/virologia , Febre/imunologia , Febre/virologia , Gastroenteropatias/imunologia , Gastroenteropatias/virologia , Hepatite/imunologia , Hepatite/virologia , Humanos , Pneumonia/imunologia , Pneumonia/virologia , Carga ViralRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects a variety of cell types in vivo. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL146, encodes an alpha-chemokine. PCR amplification and sequencing of this gene from serial samples obtained from transplant recipients and samples from infants with suspected congenital HCMV infection, revealed that UL146 is a hypervariable gene in vivo. However, genetic changes were highly conserved in individuals and in renal transplant recipients multiple genotypes of UL146 were present. The majority of strains characterized maintained the conserved ELRCXC motif present in the Toledo strain of HCMV. These results provide further evidence that AD169 does not represent the authentic virus in vivo and although Towne and Toledo are more representative, major genetic differences still exist. Mixed populations of HCMV strains occur in vivo so cloning of these strains is essential if an authentic genotype is to be defined.
Assuntos
Quimiocinas CXC/genética , Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Variação Genética , Proteínas Virais/genética , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , DNA Viral/genética , Humanos , Recém-Nascido , Filogenia , TransplanteRESUMO
The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1-UL11, UL105-UL112 and UL120-UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.
Assuntos
Citomegalovirus/genética , Genes Virais , Sequência de Aminoácidos , Quimiocinas CXC/genética , Variação Genética , Genoma Viral , Humanos , Dados de Sequência Molecular , Mutação , Filogenia , Alinhamento de Sequência , Proteínas Virais/genéticaRESUMO
Forty-eight patients who provided 2 consecutive blood samples that tested positive for cytomegalovirus DNA by polymerase chain reaction (PCR) were randomized to receive either full-dose ganciclovir (5 mg/kg intravenously [iv] twice daily) or half-dose ganciclovir (5 mg/kg iv once daily) plus half-dose foscarnet (90 mg/kg iv once daily) for 14 days. In the ganciclovir arm, 17 (71%) of 24 patients reached the primary end point of being CMV negative by PCR within 14 days of initiation of therapy, compared with 12 (50%) of 24 patients in the ganciclovir-plus-foscarnet arm (P = .12). Toxicity was greater in the combination-therapy arm. In patients who failed to reach the primary end point, baseline virus load was 0.77 log10 higher, the replication rate before therapy was faster (1.5 vs. 2.7 days), and the viral decay rate was slower (2.9 vs. 1.1 days) after therapy. Bivariable logistic regression models identified baseline virus load, bone-marrow transplantation, and doubling time and half-life of decay as the major factors affecting response to therapy within 14 days. This study did not support a synergistic effect of ganciclovir plus foscarnet in vivo.
Assuntos
Antivirais/uso terapêutico , Transplante de Medula Óssea/efeitos adversos , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/crescimento & desenvolvimento , Foscarnet/uso terapêutico , Ganciclovir/uso terapêutico , Transplante de Rim/efeitos adversos , Transplante de Fígado/efeitos adversos , Adolescente , Adulto , Idoso , Antivirais/efeitos adversos , Criança , Pré-Escolar , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , DNA Viral/química , DNA Viral/genética , Quimioterapia Combinada , Feminino , Foscarnet/efeitos adversos , Ganciclovir/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Carga ViralRESUMO
Two novel spliced genes (UL131A and UL128) flanking UL130 were predicted from sequence comparisons between human cytomegalovirus (HCMV) and its closest known relative, chimpanzee cytomegalovirus (CCMV), and the splicing patterns were confirmed by mRNA mapping experiments. Both genes were transcribed with late kinetics and shared a polyadenylation site. Comparisons with wild-type HCMV in infected human tissues showed that three of five isolates passaged in cell culture contained disruptions of UL128, one was frameshifted in UL131A and one exhibited a deletion affecting UL131A and UL130. CCMV and the Colburn strain of simian cytomegalovirus, which have been passaged in cell culture, also exhibit disruptions of UL128. These observations indicate that expression of either one of UL128 and UL131A is deleterious to growth of primate cytomegaloviruses in cell culture. Although the functions of these genes are unknown, sequence comparisons suggest that UL128 encodes a beta-chemokine.
Assuntos
Citomegalovirus/genética , Splicing de RNA , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Citomegalovirus/metabolismo , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
A significant proportion of the human cytomegalovirus (HCMV) genome comprises 12 multigene families that probably arose by gene duplication. One, the RL11 family, contains 12 members, most of which are predicted to encode membrane glycoproteins. Comparisons of sequences near the left end of the genome in several HCMV strains revealed two adjacent open reading frames that potentially encode related proteins: RL6, which is hypervariable, and RL5A, which has not been recognized previously. These genes potentially encode a domain that is the hallmark of proteins encoded by the RL11 family, and thus constitute two new members. A homologous domain is also present in a subset of human adenovirus E3 membrane glycoproteins. Evolution of genes specifying the shared domain in cytomegaloviruses and adenoviruses is characterized by extensive divergence, gene duplication and selective sequence loss. These features prompt speculation about the roles of these genes in the two virus families.
Assuntos
Proteínas E3 de Adenovirus/genética , Adenovírus Humanos/genética , Citomegalovirus/genética , Genes Virais , Adenovírus Humanos/química , Sequência de Aminoácidos , Sequência de Bases , Citomegalovirus/química , DNA Viral , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The limitations of current therapies for human cytomegalovirus (HCMV) coupled with the continued impact of HCMV disease in the immunocompromised host are the driving force for the development of new drugs against HCMV. This review predominantly focuses on new non-DNA polymerase inhibitors of HCMV replication. Drugs such as tomeglovir (BAY-384766), 2-bromo-5,6-dichloro-1beta-D-ribofuranosyl benzimidazole (BDCRB) and GW-275175X act as inhibitors of the terminase complex that is involved in cleavage and packaging of the unit length DNA into the capsids. Although the viral protein kinase UL97 has been exploited as an activator of ganciclovir and its prodrug valganciclovir, a new inhibitor maribavir (benzamidavir) has been shown to be a highly potent inhibitor of this enzyme. Many of these compounds have undergone successful phase I clinical trials. There are other compounds which have been identified through drug-screening but are at the earlier stages of development.
Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Antivirais/química , Ensaios Clínicos Fase I como Assunto , Desenho de Fármacos , Farmacorresistência Viral , Humanos , Resultado do TratamentoRESUMO
Human cytomegalovirus (HCMV) can infect both HCMV-naive and -experienced transplant patients. In this study, the growth rate of HCMV in HCMV-naive hosts (1.82 units/day; 95% confidence interval [CI], 1.44-2.56 units/day) was shown to be significantly faster than the growth rate of virus in HCMV-experienced hosts undergoing recurrent infection (0.61 units/day; 95% CI, 0.55-0.7 units/day; P<.0001). The basic reproductive number (R(0)) for HCMV-naive liver transplant patients was 15.1 (95% CI, 8.9-44) but was only 2.4 (95% CI, 2.35-2.8) for HCMV-experienced transplant recipients, corresponding to an anti-HCMV immune efficacy of approximately 84%, despite immunosuppressive therapy. The R(0) values suggest that an anti-HCMV drug or vaccine with an efficacy of >93% (95% CI, 89%-98%) is required to eliminate viral growth during infection of HCMV-naive liver transplant recipients, whereas lower efficacy levels are sufficient to reduce the R(0) value to <1 in hosts with prior HCMV immunity.