Human cytomegalovirus reactivation from latency: validation of a “switch” model in vitro

Abstract

BackgroundHuman cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in ‘at-risk’ categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies.Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model.MethodsTHP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes.For viral progeny analysis experiments, the Towne strain was also used.ResultsOur results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i.ConclusionsOur data demonstrate the effectiveness of THP-1 cells as a “switch” model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0634-z) contains supplementary material, which is available to authorized users.

Alternative promoters drive human cytomegalovirus reactivation from latency

“…We measured the accumulation of IE1 and IE2 proteins and the expression of UL123 and UL122 transcripts during experimental latency and reactivation in the monocytic THP-1 cell line. THP-1 cells are an established model for studying HCMV latency and reactivation (16)(17)(18)(19). While the THP-1 cell model does not recapitulate all aspects of latency, such as the robust production of progeny virus, the THP-1 cell line offers the strength of a synchronous reactivation.…”

“…Cells were infected with HCMV (TB40/E strain) and allowed to establish latency for 5 d post infection (dpi). To induce reactivation, latently infected cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which promotes monocyte-to-macrophage differentiation and triggers viral reactivation (16,17). IE1 and IE2 proteins were detected immediately after infection but decreased to levels below the limit of detection between 2 and 5 dpi ( Fig.…”

Alternative Promoters Drive Human Cytomegalovirus Reactivation from Latency

“…To determine if the MIEP does in fact drive the accumulation of IE1 and IE2 mRNAs during reactivation from latency, we measured the expression of IE1 and IE2 transcripts during experimental latency and reactivation in the monocytic THP-1 cell line. THP-1 cells are an established model for studying HCMV latency and reactivation (18)(19)(20) in which reactivation can be synchronized. Cells were infected with HCMV (TB40/E strain) and allowed to establish latency for 5 days post infection (dpi).…”

Reactivation of cytomegalovirus infection in patients being in critical state

“…При иммуно-супрессии, а также во время беременности пер-вичная ЦМВИ может сопровождаться чрезмерно активной репликацией вируса с поражением мно-гих типов клеток. Это приводит к серьезным по-следствиям и, в конечном итоге, может не только сопровождаться тяжелыми клиническими прояв-лениями, но и привести к смерти [12].…”

“…Диффе-ренцировка CD34+ клеток в макрофаги или ден-дритные клетки приводит к экспрессии вирусных генов, репликации вирусной ДНК и продукции вируса de novo. В настоящее время установлено, что маркером реактивации ЦМВ является индук-ция экспрессии гена IE, однако возобновление экспрессии гена IE является лишь первым шагом в каскаде событий, которые необходимы для про-дуктивной реактивации репликации вируса [12]. Получены также данные, указывающие на то, что во время латентной инфекции продуцируется ряд вирусных микроРНК, некоторые из которых могут участвовать в манипулировании миелоидной диф-ференцировкой, хотя окончательно их функции не установлены [13].…”