| Abstract |
Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a
significant pathogen that causes medically severe diseases in
immunocompromised individuals and in congenitally infected neonates. RhoB
belongs to the family of Rho GTPases, which regulates diverse cellular
processes. Rho proteins are implicated in the entry and egress from the host
cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least
studied in this regard. Here, we studied the role of RhoB GTPase during
HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells
showed that RhoB was translocated to the assembly complex/compartment
(AC) of HCMV, a cytoplasmic zone in infected cells where many viral
structural proteins are known to accumulate and assembly of new virions
takes place. Furthermore, RhoB was localized at the AC even when the
expression of the late HCMV AC proteins was inhibited. At the very late
stages of infection, cellular projections were formed containing RhoB and
HCMV virions, potentially contributing to the successful viral spread.
Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a
significant reduction of the virus titer and could also affect the accumulation of
AC viral proteins at this subcellular compartment. RhoB knockdown also
affected actin fibers' structure. Actin reorganization was observed at late
stages of infection originating from the viral AC and surrounding the cellular
projections, implying a potential interplay between RhoB and actin during
HCMV assembly and egress. Last but not least, it was found that other Rho
proteins localize at the HCMV AC, which exhibits an interesting configuration
among neighboring cells. In conclusion, our results demonstrate for the first
time that RhoB is a constituent of the viral AC and is required for HCMV
productive infection and that other Rho proteins are also implicated in the late
stages of HCMV lytic infection.
MicroRNAs (miRNAs) are small RNAs that based on their
complementarity to mRNAs, bind to them and affect, mostly negatively, their
expression. MiRNAs are expressed in a variety of organisms, such as plants,
humans and many viruses, including HCMV. Both cellular and viral miRNAs
can regulate cellular as well as viral transcripts, and in this way they can affect
the course of the infection. In this study, the effect of HCMV on the
expression of cellular miRNAs was investigated throughout the course of the
lytic infection of human fibroblasts. Using miRNA microarrays, 26 differentially
expressed miRNAs were identified. From the above miRNAs, 14 exhibited
increase and were further divided into four groups based on the time pattern
of their change, and 12 exhibited decrease and were further divided into 3
groups. Eight of the above 26 HCMV affected miRNAs, had a significant effect
on the progression of the lytic infection of the virus, since the reversion of their
virus induced change, resulted in the significant reduction of the virus titer.
These 8 miRNAs were further studied with bioinformatics and their predicted
cellular targets were identified. Among them, the most likely targets were
selected and subsequently these predictions were validated with luciferase
and Real-Time PCR assays. The miRNAs and their targets were associated
in two networks, in which a coordinated time change of the expression of the
miRNAs and the mRNAs of their targets was observed. Three miRNAs were
included in the first network, the inflammatory network, and the remainder 5
miRNAs were included in the second network, the chromatin network. These
two networks seem to play an important role in the outcome of the infection,
since the deregulation of even individual miRNAs that participate in them, had
an important negative effect on the expression of many viral genes and on the
viral yield.
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