Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells

ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Luwanika Mlera, Danielle K. Offerdahl, Craig Martens, Stephen F. Porcella, Wessam Melik, Marshall E. Bloom
Formato: article
Lenguaje:EN
Publicado: American Society for Microbiology 2015
Materias:
Acceso en línea:https://doaj.org/article/153475668fb544a18940f4a7d55a880d
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:153475668fb544a18940f4a7d55a880d
record_format dspace
spelling oai:doaj.org-article:153475668fb544a18940f4a7d55a880d2021-11-15T15:49:02ZDevelopment of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells10.1128/mBio.00614-152150-7511https://doaj.org/article/153475668fb544a18940f4a7d55a880d2015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00614-15https://doaj.org/toc/2150-7511ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. IMPORTANCE Tick-borne flaviviruses are significant infectious agents that cause serious disease and death in humans worldwide. Infections are characterized by severe neurological symptoms, such as meningitis and encephalitis. A high percentage of people who get infected and recuperate from the acute phase of infection continue to suffer from chronic debilitating neurological sequelae, most likely as a result of nervous tissue damage, viral persistence, or both. However, little is known about mechanisms of viral persistence. Therefore, we undertook studies to investigate the persistence of Langat virus, a member of the tick-borne flavivirus group, in a mammalian cell line. Using next-generation sequencing, we determined that defective viral genomes do not play a role in the initiation of persistence, but their occurrence seems to be nonstochastic and could play a role in the maintenance of viral persistence via the expression of a novel envelope-NS1 fusion protein.Luwanika MleraDanielle K. OfferdahlCraig MartensStephen F. PorcellaWessam MelikMarshall E. BloomAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution DOAJ
collection DOAJ
language EN
topic Microbiology
QR1-502
spellingShingle Microbiology
QR1-502
Luwanika Mlera
Danielle K. Offerdahl
Craig Martens
Stephen F. Porcella
Wessam Melik
Marshall E. Bloom
Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
description ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. IMPORTANCE Tick-borne flaviviruses are significant infectious agents that cause serious disease and death in humans worldwide. Infections are characterized by severe neurological symptoms, such as meningitis and encephalitis. A high percentage of people who get infected and recuperate from the acute phase of infection continue to suffer from chronic debilitating neurological sequelae, most likely as a result of nervous tissue damage, viral persistence, or both. However, little is known about mechanisms of viral persistence. Therefore, we undertook studies to investigate the persistence of Langat virus, a member of the tick-borne flavivirus group, in a mammalian cell line. Using next-generation sequencing, we determined that defective viral genomes do not play a role in the initiation of persistence, but their occurrence seems to be nonstochastic and could play a role in the maintenance of viral persistence via the expression of a novel envelope-NS1 fusion protein.
format article
author Luwanika Mlera
Danielle K. Offerdahl
Craig Martens
Stephen F. Porcella
Wessam Melik
Marshall E. Bloom
author_facet Luwanika Mlera
Danielle K. Offerdahl
Craig Martens
Stephen F. Porcella
Wessam Melik
Marshall E. Bloom
author_sort Luwanika Mlera
title Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
title_short Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
title_full Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
title_fullStr Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
title_full_unstemmed Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
title_sort development of a model system for tick-borne flavivirus persistence in hek 293t cells
publisher American Society for Microbiology
publishDate 2015
url https://doaj.org/article/153475668fb544a18940f4a7d55a880d
work_keys_str_mv AT luwanikamlera developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
AT daniellekofferdahl developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
AT craigmartens developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
AT stephenfporcella developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
AT wessammelik developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
AT marshallebloom developmentofamodelsystemfortickborneflaviviruspersistenceinhek293tcells
_version_ 1718427502968307712