From the
A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P)
of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one
containing the amino terminus of P fused to the DNA-binding domain of
the yeast transactivator, GAL4, and the other containing the amino
terminus of NP fused to the herpesvirus transactivator, VP16, were
transfected in COS-1 cells along with a chloramphenicol
acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding
sites. A specific and high-affinity interaction between NP and P was
observed as measured by the activation of the CAT gene. Mapping of the
domains in P (603 amino acids) involved in the association with NP
revealed that NH
The ribonucleoprotein (RNP)
For
many paramyxoviruses, including HPIV-3, the P mRNA has the capacity to
encode multiple proteins
(1, 2) . For HPIV-3, a protein,
designated P/D, is synthesized, in addition to the wild-type P, by an
RNA editing mechanism by incorporating Gly residues at a specific
site
(10) . A basic protein, designated C, is synthesized by
translation of an alternate +1 open reading frame of the P
mRNA
(11) . The precise functions of these proteins and their
interactions, if any, with L, P, and NP during the life cycle of the
virus remain to be determined.
To gain insight into the replication
process of HPIV-3, we have studied the formation of the NP
pGALP (HPIV-3) was constructed by amplifying the entire P gene of
HPIV-3 by using pPET3aP as template and the oligonucleotide primers
containing either a BamHI site (the 5` primer) or a
SacI site (the 3` primer). The purified and digested 1.8-kb
PCR product was ligated with BamHI/SacI-digested
pGAL4. pGALP/D was made similarly except that pPG7
(10) was used
as a template. The P mutant plasmids were constructed via PCR using
plasmid pPET3aP as template and oligonucleotide primers that spanned
the portion of the P gene of interest and contained either a
BamHI site or SacI site. To ensure that the fused P
is in frame, two extra nucleotides were added between the
BamHI site and the first codon of P at the 5` primer. The
linker joining GAL4 and all P gene sequences encodes the following
peptide: Pro-Glu-Phe-Pro-Gly-Ile-Leu.
pVPNP was made by amplifying
the entire NP gene of HPIV-3 by using pPET3aNP as template and the
oligonucleotide primers containing either an EcoRI site (the
5` primer) or a ClaI site (the 3` primer). The purified and
digested 1.6-kb PCR product was ligated with
EcoRI/ClaI-digested pVP16. The mutant pVPNP plasmids
were also made by PCR using plasmid pPET3aNP as a template and
oligonucleotide primers that spanned the portion of the NP gene of
interest and contained either an EcoRI or a ClaI
site. The linker joining all VP16 and NP sequences encodes the
following peptide: Glu-Phe-Ala.
pGALC was made by amplifying the
entire C gene of HPIV-3 by using pPET3aC (obtained by inserting a
full-length C gene into pPET3a plasmid) as template and the
oligonucleotide primers containing either a BamHI site (5`
primer) or a SacI site (3` primer). The purified and digested
0.6-kb PCR product was ligated with
BamHI/SacI-digested pGAL4. The linker joining the
GAL4 and C sequences encodes the same peptide as is present between the
GAL4 and P sequences.
Similarly, pVPC was constructed by amplifying
the entire C gene of HPIV-3 by using pPET3aC as template and the
oligonucleotide primers containing either an XbaI site (5`
primer) or a ClaI site (3` primer). To create an XbaI
site in the pVP16 vector, the pVP16 was digested with EcoRI,
filled with Klenow, fused with an 8-mer linker containing an
XbaI site, and finally digested with XbaI and
ClaI. The purified and digested 0.6-kb PCR product was ligated
with the corresponding XbaI/ClaI-digested pVP16. The
linker joining the Vp16 and C sequences encodes the following peptide:
Glu-Phe-Ser-Arg.
All plasmid constructs were confirmed by
restriction enzyme digestion and by dideoxy sequencing across each
junction region and mutant region.
Fig. 1
shows the results of the
three-plasmid transfections. When the reporter plasmid was transfected
(CAT alone), when pGALP (P
Next, we studied the requirement of
the NH
It is becoming increasingly clear that P of negative strand
RNA viruses, with linear single strand RNA as the genetic material, is
an important auxiliary protein with direct roles in transcription and
replication of the virus
(1, 5, 20) . P acts as a
transcription factor when it complexes with L; phosphorylation plays an
important role in this process in the VSV
system
(21, 22, 23) . P also complexes with NP to
possibly keep the latter protein in a replication-competent form
(8) and facilitates the L
In the present
studies, using a two-hybrid system, we have demonstrated that NP and P
of HPIV-3 interact very efficiently in vivo (Fig. 1).
The interesting observation is that deletion mapping of P (603 amino
acids) revealed the presence of a highly interactive domain spanning
amino acid residues 63-403 (340 amino acids). An
NH
The in vivo interaction studies also demonstrated that P/D, although it
contains NH
Our results in the HPIV-3 system show that both
NH
Finally, it is
important to note that the various mutant proteins, both P and NP, used
in the experiments have the potential to undergo conformational changes
caused by specific mutations. Thus, the actual interactive site(s)
within the protein may not be located within the deleted domains. The
change of conformation of the mutant proteins may directly effect the
interacting site(s) present elsewhere in the proteins. Detailed studies
along these lines would certainly shed light to understand the role of
the P
We thank Drs. Bishnu P. De, Tapas Das, and Adrienne M.
Takacs for valuable comments on the manuscript and advice throughout
the project.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal 40 and COOH-terminal 20 amino
acids are important for such association. Interestingly, a stretch of
NH
-terminal amino acids as short as 63-403 interacted
with NP more than the wild type, reaching greater than 2.5-fold as
measured by the CAT assay. These results suggest that a domain is
present in P that negatively regulates its interaction with NP.
Deletion of NH
-terminal 40 and COOH-terminal 160 amino
acids of NP reduced the CAT activity by more than 95%. These results
underscore the important differences between negative strand RNA
viruses with respect to interactions between these two viral proteins
involved in gene expression.
(
)
complexes of
human parainfluenza virus type 3 (HPIV-3) contain a single negative
strand genome RNA (15.4 kb) complexed with at least three viral
proteins
(1, 2) . The nucleocapsid protein (NP) is the
most abundant component that encapsidates the genomic RNA to form the
NP-RNA template and maintains the structural integrity and the template
function of the RNA genome. The other two proteins associated with the
NP-RNA template constitute the RNA polymerase complex, which consists
of the large protein (L) and the phosphoprotein (P). L (251 kDa) is
likely to be the RNA polymerase, whereas P (90 kDa) is an auxiliary
protein essential for the function of L
(3) . The three
RNP-associated proteins play important roles in the life cycle of the
virus. During the transcriptive phase, in vivo or in
vitro, the L
P complex interacts with the NP-RNA template to
transcribe the genomic RNA into six distinct mRNAs
(4) . On the
other hand, during the replication step in vivo, NP forms a
soluble complex with P, and the resulting NP
P complex interacts
with the transcribing RNP to switch transcription reaction to
replication
(5) . The requirements of the formation of both the
L
P complex and the NP
P complex in transcription and
replication, respectively, have been shown in vitro and in
vivo for vesicular stomatitis virus (VSV), a prototype negative
strand RNA virus
(6, 7, 8) . Recently, this has
also been demonstrated in Sendai virus, a paramyxovirus, by expression
of the recombinant proteins in the cell
(5, 9) . The
precise mechanism and the roles played by the respective complexes in
the switch from transcription to replication remain unclear.
P
complex, which is the essential component involved in the switch from
transcription to replication. We used a modified two-hybrid system
(12) to study the nature of the interaction between these two
proteins in an in vivo context as well as map the interacting
domains. Using a GAL4/VP16-based three-plasmid transfection system we
have been able to identify the interacting domains of N and P of
VSV
(13) . Here we report that NP and P of HPIV-3 interact very
strongly in vivo, and by mutational studies, we identified the
interacting domains. Additionally, a domain within P has been
characterized that seems to act as a negative regulator in its
interaction with NP.
Plasmids
pGAL4, originally called pSG424, which
encodes the DNA-binding domain of GAL4 under the SV40 ori/early
promoter control, was provided by M. Ptashne (Harvard
University)
(14) . pVP16, originally named pAASVVP16, which
encodes the VP16 transactivating domain of herpesvirus under the SV40
ori/early promoter control, was provided by H. Vasavada (A. Miles
Co.)
(15) . pGALVP16, originally named pSGVP490, which
encodes GAL4 DNA binding domain fused with VP16 transactivating domain
was provided by M. Ptashne and has been described
elsewhere
(16) . The reporter plasmid, pG5B-CAT, which contains
five copies of the GAL4 binding site, the E1b TATA promoter, and the
CAT gene, was also provided by M. Ptashne (Harvard University) and has
been described previously
(17) . The internal control plasmid,
pRSV-
-gal, which contains the
-galactosidase gene under Rous
sarcoma virus promoter control, was obtained from the Promega Company.
pVPN of VSV and pGALP of VSV were described previously (13, 18).
Plasmid Transfection and CAT Assay
COS-1 cells
were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 2
mM glutamine, 100 units/ml of penicillin, and 100 units/ml of
streptomycin. For transfection, 0.5 µg of pRSV--gal, 0.8
µg of the test and reporter plasmids were mixed with 20 µg of
Lipofectamine (Life Technologies, Inc.) in OPTI-MEM medium (Life
Technologies, Inc.) for at least 15 min and then added to a 60
35-mm dish containing 5
10
cells subcultured the
previous day. After 6 h the transfection solution was aspirated away,
and the cells were grown in 4 ml of Dulbecco's modified
Eagle's medium with serum for 48 h. Cells were harvested, and 2
µg of cell extract was assayed for CAT activity (19). After
autoradiography of the separated acetylated chloramphenicol forms, the
spots were quantitated using a PhosphorImager (Molecular Dynamics). 10
µg of cell extract was assayed for
-galactosidase
activity
(19) .
Western Blot Analyses of Expressed Proteins
A
rabbit polyclonal antibody to GAL4 (amino acids 1-147) was
provided by M. Ptashne (Harvard University). A rabbit polyclonal
antibody to HPIV-3 was kindly provided by Ranjit Ray (St. Louis
University). The expression levels of all the VPNP (wild-type as well
as mutant) chimeras in the transfected cell extracts were determined by
immunoblotting with the antibody to HPIV-3 using gene screen membrane
(DuPont) following the manufacturer's protocol. The expression
levels of GALP and GALC chimeras in the transfected cell extracts were
similarly assayed by the antibody against GAL4.
Association of the HPIV-3 NP and P in Vivo
A
GAL4/VP16-based three-plasmid system was used to study the association
of NP and P in vivo. Plasmids were constructed that encoded
the following fused proteins: the amino terminus of P of HPIV-3 fused
to the GAL4 DNA-binding region and the amino terminus of NP of HPIV-3
fused to the VP16 transactivating domain. When these two plasmids along
with the CAT reporter plasmid were co-transfected into COS-1 cells, any
association of NP and P in vivo would activate transcription
of the CAT gene via the VP16 transactivating region. This could then be
assayed by the conversion of [C]chloramphenicol
to its acetylated forms.
) was
transfected with pVP16, or when pVPNP
(NP
) was transfected with pGAL4, no CAT
activity was detected. pGALVP16 (GALVP), which produced a high level of
CAT activity, served as positive control. These results indicate that
either NP fused to VP16 or P fused to GAL4 alone cannot activate the
CAT gene by binding to the promoter or to any proteins associated with
the promoter. However, when both pGALP and pVPNP were co-transfected
with the reporter plasmid there was an extremely high level of CAT gene
expression, which was shown by more than 90% conversion of
[
C]chloramphenicol to its acetylated forms. This
result demonstrates the high affinity interaction between NP and P
in vivo. Since the CAT activity from NP and P co-transfection
is so high, we titrated the cell extract used in the assay to determine
the amount that would result in CAT activity within the linear range.
Fig. 2
shows that cell extracts containing as little as 5.0 µg
of protein resulted in almost complete conversion of
[
C]chloramphenicol to its acetylated forms.
Depending on the individual cell extract, only 1.0-2.0 µg of
protein equivalent of cell extract was needed to obtain CAT activity
within the linear range. This concentration of cell extract was used in
all subsequent CAT assays. Each fraction was also tested for
-galactosidase activity, which measured the efficiency of
transfection. The CAT activity in each fraction was calculated based
upon
-galactosidase activity. In a separate series of experiments
(data not shown) the expression levels of P and NP chimeras were
measured by Western blot analyses (see ``Materials and
Methods''). The extent of protein synthesis in each reaction was
similar.
Figure 1:
The
association of NP and P of HPIV-3 in vivo. COS-1 cells were
co-transfected with the indicated plasmids and the reporter plasmid.
The resulting cell extracts were then assayed for CAT activity as
described under ``Materials and Methods.'' 2.0 µg of cell
extract was used for each reaction. The percentage of acetylation
(% Acetyl.) was quantitated by using a PhosphorImager and
standardized by -galactosidase activity. P, pGALP of
HPIV-3; NP, pVPNP of HPIV-3; P, pGALP of VSV;
NV, pVPN of VSV.
Figure 2:
Titration of CAT activity from the
transfected cell extract. COS-1 cells were co-transfected with pGALP,
pVPNP, and the reporter plasmid. Aliquots of cell extracts as indicated
were used for CAT assay as described in the text. CE, cell
extract.
To confirm that the CAT activity is due to a specific NP
and P interaction, the HPIV-3 P was replaced with P of VSV
(P<itinf;V). Similarly, HPIV-3 NP was replaced by
nucleocapsid protein(N) of VSV (N ). When
the heterologous plasmids were co-transfected, no CAT activity was
detected in either case. As expected, VSV P- and N-containing
appropriate plasmids produced high CAT activity
(13) . These
results clearly show that NP of HPIV-3 does not interact with P of VSV,
or vice versa. Thus, the interaction between NP and P is highly
specific for HPIV-3.
Domains of P Involved in Its Interaction with
NP
Next, we tested different P mutants to investigate the
domains of P involved in the interaction with NP. As shown in
Fig. 3
, deletion of the COOH-terminal 10 amino acids (10C)
reduced the CAT activity by 40% while
20C reduced the CAT activity
by 70%, suggesting that the COOH terminus of P is involved in its
association with NP. Interestingly, further deletions of amino acids
from the COOH terminus, e.g.
40C and
100C and so on,
resulted in a gradual increase in CAT activity. The highest CAT
activity (more than 250% of the wild type) resulted when 410 amino
acids were removed from the COOH terminus (
410C), leaving only an
NH
-terminal 193-amino acid fragment. Further deletions from
the COOH-terminal end, i.e.
510C and
540C, resulted
in a slight decrease in interaction, but the truncated proteins
continued to interact strongly. However, the deletion mutant
570C
lost completely its capacity to interact with NP. Thus, an
NH
-terminal P fragment as short as 63 amino acids long
interacted with the NP more strongly (greater than 2-fold) than the
wild type. The largest NH
-terminal fragment that interacted
more than the wild type is
200C (Fig. 3), i.e. 403
amino acids long. These data strongly suggest that a domain is present
in P, which resides between amino acids 63 and 403, that negatively
regulates its interaction with NP.
Figure 3:
Domains of P involved in the association
with NP. A, schematic representation of P showing the
COOH-terminal deletion mutants of the 603-amino acid P fused to the
GAL4 DNA-binding region. The number of amino acids deleted from the
COOH-terminal end of P is indicated. B, CAT activity is shown
from the co-transfection of pGALP mutants with pVPNP and the reporter
plasmid. 2.0 µg of cell extract was used in each reaction. Average
CAT activity from at least three experiments of each P mutant was
determined by CAT assay and expressed as a percentage of that of
wild-type P, representing 100% CAT activity.
To further confirm that the
removal of such a regulatory domain from P indeed increases the
association of P with NP, we constructed a plasmid (PAB, Fig. 4)
that contains 230 NH-terminal amino acids linked in-frame
with the COOH-terminal 20 amino acids of P. As shown in Fig. 5,
the PAB interacts with NP as efficiently as the P
350C (250 and
240% of the wild type, respectively), confirming that the
NH
-terminal one-third of P interacts more strongly (more
than 2.5-fold) than the wild-type P. The deleted portion of P thus
appears to act as the negative regulatory domain in its association
with NP.
Figure 4:
Association of NP with internally
deleted P and P/D. A, schematic representation is shown of the
various P mutants fused to the GAL4 DNA-binding region. The number of
amino acids deleted from P is described under ``Results.''
B, CAT activity of the co-transfection of pGALP mutants with
pVPNP and the reporter plasmid is shown. 2.0 µg of cell extract was
used for each reaction. Average CAT activity from at least three
experiments was determined by CAT assay and expressed by a percentage
of that of wild-type P, representing 100% CAT
activity.
Figure 5:
The
requirement of the NH terminus of P for the association
with NP. A, schematic representation of
NH
-terminal deletion mutants of P fused to the GAL4
DNA-binding region. The number of amino acids deleted from the
NH
-terminal end of P is indicated. B, CAT activity
of the co-transfection of pGALP mutants with pVPNP and the reporter
plasmid is shown. 2.0 µg of cell extract was used in each reaction.
Average CAT activity from at least three experiments of each P mutant
was determined by CAT assay and expressed as a percentage of that of
wild-type P, representing 100% CAT activity.
Fig. 4
also shows that the P/D protein, which is
synthesized by an RNA editing mechanism
(10) although it
contains the NH-terminal 241 amino acids, interacts poorly
with NP compared with P
350C or PAB and even less than the
wild-type P (70% of the wild type). Thus, it seems that the
frameshifted COOH-terminal portion of the P/D down-modulates the
interaction between P/D and NP.
-terminal region of P for its association with NP. As
shown in Fig. 5, removal of 20 amino acids from the NH
terminus (
20N) decreases the CAT activity by more than 80%.
Further removal of amino acids (
40N and
80N) resulted in
total abrogation of CAT activity. These data, coupled with the
COOH-terminal deletion data (Fig. 3), indicate that both ends of
P are needed for its interaction with NP. However, the
NH
-terminal domain seems to be more critical than the
COOH-terminal domain since further deletion from the latter end
increases its interaction with NP.
Interaction of C with NP or P
The same
three-plasmid system was used to study the possible interaction between
NP and C or P and C in vivo. Plasmids were constructed that
encode the C protein fused to either the GAL4 DNA-binding region or the
VP16 transactivating domain. As shown in Fig. 6, when pGALC or
pVPC were co-transfected with VPNP or GALP, respectively, along with
reporter plasmid, no CAT activity was detected, even when 5-fold excess
of the cell extracts compared with pGALP and pVPNP were used. These
results indicate that the C protein of HPIV-3 does not interact with P
or NP of HPIV-3 in this system.
Figure 6:
Association of NPC or P
C of
HPIV-3 in vivo. COS-1 cells were co-transfected with the
indicated plasmids and the reporter plasmid. The resulting cell
extracts were assayed for CAT activity as described under
``Materials and Methods.'' P, pGALP of HPIV-3;
NP, pVPNP of HPIV-3; VPC, VP16 fused to C; GALC, Gal
fused to C.
All P mutants, P/D, and C were
tested by Western blot analyses to confirm their extent of expression.
The expression levels are quite consistent, comparable with that of the
wild-type P, and easily detected by Western blot analyses (data not
shown).
Domains of NP Involved in Its Interaction with P in
Vivo
Finally, we studied the domain of NP responsible for
interaction with P. By systematic NH- and COOH-terminal
deletions of NP, we made several VPNP mutant chimeras. As shown in
Fig. 7
, deletions of the COOH-terminal 20 (
20C) and 80
(
80C) amino acids did not reduce CAT activity significantly.
However, deletion of an additional 80 amino acids (
160C) totally
abrogated the interaction. Similarly, deletion of the
NH
-terminal 20 amino acids (
20N) did not reduce the
CAT activity, but removal of 40 (
40N) and 80 (
80N) amino
acids from the NH
terminus reduced the CAT activity by more
than 95%. These results indicate that both ends of NP are needed for P
interaction but that the NH
terminus of NP seems to be
critical for its association with P.
Figure 7:
Domain of NP required for the association
with P in vivo.A, schematic representation of the NP
protein showing the systematic NH- and COOH-terminal
deletion mutants fused to the VP16 transactivating domain. The number
of amino acids deleted from both ends of the 515-amino acid NP are
indicated. B, CAT activity from the co-transfection of pVPNP
mutants with pGALP and the reporter plasmid. 2.0 µg of cell extract
was used in each reaction. Average CAT activity from at least three
different experiments of each NP mutant was determined by CAT assay and
expressed as a percentage of that of wild-type NP, representing 100%
CAT activity.
All NP mutants were tested by
Western blot analysis. The expression levels are consistent with that
of the wild-type NP (data not shown).
P complex to switch from
transcription to replication. The precise mechanism by which these
complexes interact with each other and the N RNA template leading to
the replication reaction remains unclear. In the well studied VSV
system
(13) , and more recently in rabies virus
(24) , both
NH
- and COOH-terminal domains of P seem to be required for
its interaction with N. In the Sendai virus, the COOH-terminal end
appears to interact with the viral RNP
(25, 26) , whereas
the NH
-terminal domain acts as a chaperone for NP during
chain assembly of genome replication
(27) . The COOH-terminal
domain of Sendai virus NP appears to be specifically involved in its
interaction with P
(28) . The domains in P involved in the
interaction with L in the VSV system appear to be located both at the
NH
- and COOH-terminal domain of
P
(29, 30) .
(
)
-terminal 63-amino acid fragment of P is capable of
interacting with NP more than two times as efficiently than the wild
type (Fig. 3). Since removal of 20 amino acids from the
NH
-terminal end of P decreased its interaction with NP to
30% (Fig. 4), it seems that the NH
-terminal domain
between amino acids 1 and 63 contains both the essential and highly
interactive domain for P. Thus, it seems that the structure of the
HPIV-3 P protein is quite unique. In the wild-type configuration P
down-regulates a highly interactive domain within the protein, which
stretches from the NH
-terminal penultimate amino acid to
403. A negative regulatory domain seems to be located within the
COOH-terminal 200-amino acid stretch, since removal of this domain
significantly increases the interaction of the remaining P with the NP.
It is interesting to note that within this domain removal of the
COOH-terminal 20 amino acids (Fig. 3) significantly decreases the
interaction with NP. Thus, the COOH-terminal domain spanning 200 amino
acids perhaps contains both negative and positive elements that
regulate its interaction with NP. The precise structure of such a
domain in the function of P remains to be elucidated. Clearly, the
presence of such an interactive/regulatory domain in P seems to be
unique for HPIV-3, since a domain with similar characteristic is not
present in the VSV P
(13) . In the prototype Sendai virus system,
two noncontiguous domains at the COOH-terminal end of P have been
identified that interact with the viral RNP
(25, 26) .
The L binding site has recently been shown to reside between the two
RNP binding domains
(20, 31) . Assuming a similar
situation exists in the HPIV-3 P, the putative regulatory domain,
interestingly, would cover both the RNP-binding and the L-binding
domains. It remains to be determined whether binding of L and RNP
within this domain could influence the interaction of the highly
reactive free NH
-terminal P protein with the soluble NP for
genome replication
(27) . It is noteworthy that in the VSV system
the NH
-terminal domain of P is also a transactivator that
can complement functionally in trans the COOH-terminal end of
P bound to the NH
-RNA template and L in
transcription
(32) . Whether the highly NP-interactive
NH
-terminal domain is similarly active in transcription in
the HPIV-3 P remains to be determined.
-terminal superinteracting domain, interacts
with NP less efficiently than the wild-type P (Fig. 4). This
clearly indicates that the frameshifted region at the COOH-terminal end
exerts a negative effect on the remaining NH
-terminal
highly NP-P interacting domain. The precise role of the P/D in the
life-cycle of the virus remains unclear. In Sendai virus, however, the
edited V protein has been shown to have a regulatory role in viral
replication
(20) , although it does not seem to interact with
L
(20, 31) . It is noteworthy that in the HPIV-3 system,
in addition to P and P/D, a preterminated NH
-terminal P
(Pt) is synthesized as a result of the editing reaction and is found to
be present in HPIV-3-infected cells
(10) . Since Pt contains the
NH
-terminal 242 amino acid residues, it is expected that it
would interact with NP more strongly than the wild-type P. An
interesting possibility is whether such protein would regulate
replication reaction by binding more tightly with NP. We have also
shown that the HPIV-3 C protein does not interact with P or NP
(Fig. 5), although it appears to be present in the purified
virion
(3) .
- and COOH-terminal ends of P are needed for its
interaction with NP; the NH
-terminal end seems to be more
important than the COOH-terminal end. These results are similar to that
for the VSV and rabies P
(13, 24) . However, the domain
of NP involved in interaction with P is quite different in HPIV-3 and
VSV. Unlike VSV, the interacting domain lies at the
NH
-terminal domain of HPIV-3 NP (Fig. 7). In the
Sendai system, using a protein-blotting technique, Homann et
al.(28) have observed an important role of the
COOH-terminal domain of NP for P interaction. Interestingly, deletion
of an internal 20 residues in the hydrophobic part of NP completely
abolished its binding to P. Thus, it seems that different negative
strand RNA viruses have evolved in a manner such that different domains
of P and NP interact with each other to carry out the precise function
of the P
NP complex in the replication reaction.
NP complex and the structure of the interacting domains of
the two proteins involved in the genome RNA replication.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.