|Virus-Associated RNA I (VA-RNAI; VAI) of adenovirus is a nontranslated
RNA molecule known to interact with several dsRNA-binding
molecules in host cells. Adenovirus lacking VA-RNAI shows reduced
replication and toxicity in normal tissues but replicates with high efficiency
in some cancer cell lines.
In this work, efficient replication of VA-RNAI-deleted adenovirus
dl331 was confirmed to be independent of cancer cells’ k-ras mutation
status, leaving the basis of its selectivity unknown.
VA-RNAI expression was necessary to prevent dsRNA-dependent
phosphorylation of eIF2α in Suit-2 and HCT116 cells, with a smaller effect
in PANC-1 and none in MiaPaCa2.
VA-RNAI was shown to suppress virus-induced autophagy in two cell
lines, whether expressed from the virus or a plasmid. Unexpectedly, the
viral protein E1A was observed to colocalise with autophagic vesicles. VARNAI
was also shown to prevent starvation-induced autophagy in
susceptible cell lines.
RNA was collected from normal NHBE cells infected with VA-RNAIdeleted
dl331 or VA-RNAI-intact control dl309 viruses. Microarray
analysis of these samples showed a significant change in gene expression as
VA-RNAI accumulated in the cells. Most notably, several genes involved in
cell cycle progression were upregulated during infection with dl331 when
compared to dl309. RT-qPCR showed a similar pattern of gene regulation in
Suit-2 cells responding to infection.
Cell cycle analysis of infected Suit-2 cells showed a transient
accumulation of cells in S-phase in response to dl309 but not dl331 at the
approximate time that VA-RNAI reached its highest level.
This work has demonstrated that VA-RNAI's ability to block eIF2α
phosphorylation inhibits the induction of autophagy after infection or
starvation. Given the newly understood importance of autophagy in cancer
pathogenesis and the responses of malignant cells to radiation and
chemotherapeutic drugs, this new function is potentially important for the
design of future oncolytic adenoviruses. However, this activity is not
sufficient to explain support for replication of VA-RNAI-deleted viruses in
cancer cell lines. Data from the microarray and cell cycle analyses suggest
that VA-RNAI may play a further role in modulating the host cell’s
replication cycle, although the mechanism for this is currently unclear.