Date Published: January 24, 2019
Publisher: Public Library of Science
Author(s): Leandro Crisostomo, Andrea Michelle Soriano, Megan Mendez, Drayson Graves, Peter Pelka, Michael Nevels.
Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.
Adenoviruses are a family of small DNA viruses that infect a variety of human and vertebrate tissues and cell types . Due to the state of the infected cell that is generally restrictive to viral replication, evolution has shaped adenoviral proteins into exquisite reprogrammers of the cell to ensure productive viral replication . Successful viral replication requires that the viral proteins are expressed in an orderly fashion. This enables the reprogramming of the infected cell without alerting the immune system to the infection, or leading to the cell instituting an antiviral defensive program that would lead to abortive infection. The adenovirus capsid is relatively small and does not allow for packaging of an extensive cohort of viral proteins to kick-start the replicative program ; rather, the virus packs a very limited number of proteins that are mostly associated with the viral genome and all proteins that carry out cellular reprogramming are expressed de novo after the viral genome is delivered to the host cell nucleus . During human adenovirus (HAdV) infection, the first gene expressed is E1A from a promoter located at the left end of the genome . Expression of E1A is thought to be governed mainly by cellular factors , although E1A is able interact with E1A-promoter bound factors and enhance its own expression [3, 4]. Upon E1A expression, other early proteins are expressed that are generally thought to be driven by E1A in coordination with the cellular transcriptional machinery, and includes the E2, E3 and E4 transcriptional units [3, 4]. Early in the infection, the Major Late Promoter (MLP) expresses only a small subset of mRNAs and is largely supressed . Consequently, expression of late proteins is low in the early phases of replication; however, this promoter becomes activated with commencement of the viral genome replication leading to high levels of late protein expression and accumulation of all late transcripts [5, 6]. Expression of late proteins is also regulated by products of the L4 gene, which activates late protein expression after initiation of viral genome replication . E1A plays a role in the regulation of expression for all early transcriptional units as well as the MLP as it has been shown to be associated with all of these promoters . In addition, viral non-coding RNAs are expressed early in infection utilizing the cellular RNA Polymerase III while all other viral transcription is performed by the cellular RNA Polymerase II . Overall, the virus carries out a highly orchestrated transcriptional program that converts a non-permissive cell into a permissive one in an efficient manner and within hours of infection.
In the present study we have used the highly sensitive digital PCR combined with probes to evaluate the temporal expression pattern of viral genes early in the infection of arrested IMR-90 cells with HAdV strain dl309, since this strain is widely used for generation of other viral mutants and vectors. We have also established the exact time of initiation of viral genome replication and its relation to viral gene expression for this strain and this cell type at a MOI of 50. Lastly, we provide a comparison table (Table 1) with earlier studies performed in HeLa cells. Therefore, our investigation provides a comprehensive picture of viral gene expression that may be of use to those that use the dl309 mutant and its derivatives for their studies.