The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in
cis
). Efficient complementation
in vivo
of replication complex formation by viral proteins provided in
trans
, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient
trans
complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in
cis
. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3C
pro
protease between 3C
pro
and 3D
pol
(3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in
trans
. Only a defect of 3D
pol
activity of the replicon could be rescued in
trans
; inactivating mutations in 2C
ATPase/hel
, 3B, and 3C
pro
of the replicon completely abrogated the
trans
-rescued replication. An intact N-terminus of the 3C
pro
domain of the 3CD
pro
provided in
trans
was essential for the
trans
-active function. By using this
trans
complementation system, a high-titer defective PV pseudovirus (PV
pv
) (>10
7
infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on
trans
complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection.
IMPORTANCE
Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in
trans
) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in
cis
) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.