RESUMEN
The broad tissue distribution and cell tropism of human cytomegalovirus indicates that the virus successfully replicates in tissues with various nutrient environments. HCMV requires and reprograms central carbon metabolism for viral replication. However, many studies focus on reprogramming of metabolism in high nutrient conditions that do not recapitulate physiological nutrient environments in the body. In this study, we investigate how HCMV successfully replicates when nutrients are suboptimal. We limited glucose following HCMV infection to determine how glucose supports virus replication and how nutrients potentially present in the physiological environment contribute to successful glucose independent HCMV replication. Glucose is required for HCMV viral genome synthesis, viral protein production and glycosylation, and virus production. However, supplement of glucose-free cultures with uridine, ribose, or UDP-GlcNAc-metabolites that support upper glycolytic branches-resulted in partially restored viral genome synthesis and subsequent partial restoration of viral protein levels. Low levels of virus production were also restored. Supplementing lower glycolysis in glucose-free cultures using pyruvate had no effect on virus replication. These results indicate nutrients that support upper glycolytic branches like the pentose phosphate pathway and hexosamine pathway can compensate for glucose during HCMV replication to support low levels of virus production. More broadly, our findings suggest that HCMV could successfully replicate in diverse metabolic niches, including those in the body with low levels of glucose, through alternative nutrient usage.
RESUMEN
Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent in vitro and in vivo data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.
Asunto(s)
Infecciones por Enterovirus , Enterovirus , Histonas , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cafeína , ADN , Daño del ADN , Enterovirus/fisiología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/metabolismo , Histonas/genética , Histonas/metabolismo , Interacciones Microbiota-Huesped , Ratones , Proteínas Virales/genética , Replicación ViralRESUMEN
The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Genoma Viral , Herpesvirus Humano 1/genética , ARN Guía de Kinetoplastida/genética , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Chlorocebus aethiops , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Endonucleasas/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Herpesvirus Humano 1/metabolismo , Recombinación Homóloga , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Selección Genética , Células Vero , Replicación ViralRESUMEN
The cleavage products from coronavirus polyproteins, known as the non-structural proteins (nsps), are believed to make up the major components of the viral replication/transcription complex. In this study, several nsps encoded by avian gammacoronavirus infectious bronchitis virus (IBV) were screened for RNA-binding activity and interaction with its RNA-dependent RNA polymerase, nsp12. Nsp2, nsp5, nsp8, nsp9 and nsp10 were found to bind to untranslated regions (UTRs), while nsp8 was confirmed to interact with nsp12. Nsp8 has been reported to interact with nsp7 and functions as a primase synthesizing RNA primers for nsp12. Further characterization revealed that nsp8-nsp12 interaction is independent of the UTRs of viral RNA, and nsp8 interacts with both the N- and C-terminal regions of nsp12. These results have prompted a proposal of how the nsp7-nsp8 complex could possibly function in tandem with nsp12, forming a highly efficient complex that could synthesize both the RNA primer and viral RNA during coronavirus infection.
Asunto(s)
Virus de la Bronquitis Infecciosa/fisiología , Multimerización de Proteína , Proteínas de Unión al ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Humanos , Unión Proteica , ARN Viral/metabolismoRESUMEN
Genome replication by the baculovirus DNA polymerase often generates errors in mononucleotide repeat (MNR) sequences due to replication slippage. This results in the inactivation of genes that affects different stages of the cell infection cycle. Here we mapped these MNRs in the 59 baculovirus genomes. We found that the MNR frequencies of baculovirus genomes are different and not correlated with the genome sizes. Although the average A/T content of baculoviruses is 58.67%, the A/T MNR frequency is significantly higher than that of the G/C MNRs. Furthermore, the A7/T7 MNRs are the most frequent of those we studied. Finally, MNR frequencies in different classes of baculovirus genes, such as immediate early genes, show differences between baculovirus genomes, suggesting that the distribution and frequency of different MNRs are unique to each baculovirus species or strain. Therefore, the results of this study can help select appropriate baculoviruses for the development of biological insecticides.