TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/22702/
Y1  - 2017///
ID  - heidok22702
TI  - Point Mutations in Nonstructural Coding Sequences of Rat 
H-1 Parvovirus and Consequences for Virus Fitness
AV  - public
A1  - Hashemi, Hamidreza
N2  - Some rodent parvoviruses including rat H-1PV virus are of particular importance for cancer
therapy because of their intrinsic oncotropism owing to strict dependence on DNA replication
machinery and other cellular factors that are provided by cells only when going through S phase
of the cell cycle, a hallmark of transformed cells. Isolation of mutant viruses that replicate more
efficiently in cancer cells has been used as a strategy to improve oncolytic properties of viruses.
Previously in our lab, a fully infectious H-1PV mutant containing a 114 nt in-frame deletion in NS
coding sequences (DelH-1PV) was analyzed by Weiss et al. This mutant exhibited key fitness
features including enhanced infectivity in vitro and stronger anti-tumor activity in vivo compared
to wt virus. Significant improvements in both early and late steps of the virus cycle were
observed including a more efficient virus binding and uptake by host cells leading to earlier viral
DNA replication, and earlier/faster nuclear export of infectious virions. In the current study, the
effects of the above-mentioned deletion on virus fitness was further investigated by introducing
point mutations in this region. The mutations clustered in the NS1/2-coding DNA sequence
corresponding to the deletion sequence in DelH-1PV. Four mutants (H1-PM-I, H1-PM-II, H1-PM-III
and H1-DM) were generated in which either NS2 (PM-II and PM-III) or NS1 and NS2(PM-I and
DM) were modified and analyzed for their fitness phenotype. Our results show that the
mutations PM-I, PM-II and DM improved some early steps of the infection cycle indicated by
more efficient cell uptake of the virus particles. Viral DNA replication was stimulated by these
mutations leading to higher production of progeny virions and better spread of the virus. To
analyze the fitness effects of NS1 mutation (Y595H) independently of NS2, NS2-null derivatives
were created. Our data showed that the NS1Y595H mutation stimulates viral DNA replication and
spread in the absence of NS2. In contrast, PM-III mutation in NS2 (L153M) compromised virus
replication and spread, indicated by a small plaque phenotype, lower yield and infectivity of the
progeny particles. However, cellular binding and uptake of the virus particles were not impaired
by this mutation. A strong cis effect of PM-III mutation on capsid production was observed at a
post-transcriptional level. Altogether our results suggest that PM-III mutation may interfere with
efficient translation of downstream open reading frame (capsid ORF) and efficient splicing of
VP1/VP2 pre-mRNA leading to decreased and imbalanced production of the capsid proteins and
consequently inefficient generation of pre-assembled capsids.
ER  -