Biological roles and functional mechanisms of arenavirus Z protein in viral replication.
ABSTRACT Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.
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ABSTRACT: Arenaviruses are important human pathogens with no FDA-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analog ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in the arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, a FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of LCMV directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces to half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live attenuated vaccine Candid#1 strain of the New World arenavirus Junin. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.Journal of General Virology 01/2013; · 3.13 Impact Factor
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ABSTRACT: Lassa virus (LASV) causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP) motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV), which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173) in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1), orthomyxo- (influenza virus PA), and bunyaviruses (La Crosse virus NL1). Although the catalytic residues (D89, E102 and K122) are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.PLoS ONE 01/2014; 9(2):e87577. · 3.53 Impact Factor
Biological Roles and Functional Mechanisms of Arenavirus Z Protein
in Viral Replication
Jialong Wang, Shamika Danzy,* Naveen Kumar,* Hinh Ly, and Yuying Liang
Department of Veterinary and Biomedical Sciences, University of Minnesota, Twin Cities, Minnesota, USA
LASV causes endemic infections in West Africa with estimated
a Junin virus vaccine, no licensed vaccine for human usage is cur-
rently available for prevention of pathogenic arenavirus infec-
portive cares. Ribavirin, a nonspecific antiviral compound, has
shown some levels of efficacy only if it is administered at an early
stage of viral infection when the symptoms are insidious (15).
Immune therapy such as the transfusion of immune plasma has
also been used to treat Argentine hemorrhagic fever (8).
Arenaviral bisegmented RNA genomes encode four proteins
that include the glycoprotein precursor GPC, the nucleoprotein
trix protein Z (2). The Z protein is a small 15-kDa RING domain
ing a matrix layer in between the lipid membranes and the NP
density layer within viral virions (17, 21). It has been reported to
proteins and glycoproteins into virus-like particles (VLPs) (18,
23). In addition, the Z protein can strongly inhibit viral RNA
synthesis (6) by directly locking the L polymerase protein in a
catalytically inactive state (12) and therefore is postulated to play
scription. Recently, the Z proteins of New World pathogenic
arenaviruses Junin, Machupo, Guanarito, and Sabia virus have
also been shown to inhibit RIG-1-dependent innate immune
Many efforts have been made to understand the molecular
mechanisms of the Z protein. It contains an N-terminal myristy-
essential for its membrane association and for self-budding activ-
ity (19, 24). The L domain(s) mediates Z budding activity by in-
everal arenaviruses, including Lassa fever virus (LASV), can
cause severe and lethal hemorrhagic fever diseases in humans.
Z-mediated inhibition of lymphocytic choriomeningitis virus
(LCMV) promoter-driven reporter RNA synthesis requires the
structural integrity of the RING domain but not the N-terminal
residues 1 to 16 or the C-terminal residues 79 to 90 (5).
cycle. By taking advantage of our recently developed reverse ge-
netics system for Pichinde virus (PICV) (14), a nonpathogenic
arenavirus that can cause Lassa fever-like symptoms in infected
guinea pigs (1, 11, 27, 28), we have undertaken an effort to exam-
ine the biological roles of each of the conserved residues and do-
mains within the Z protein. Our results have demonstrated that
most of the conserved residues are absolutely essential for virus
replication, including the G2 residue, the zinc-binding C and H
the C terminus of the protein. The C-terminal L domain can tol-
reduced viral infectivity. Additional functional characterizations
of the LASV and PICV Z proteins have provided some important
insights into the molecular mechanisms of Z protein in support-
ing infectious virus replication.
Received 15 February 2012 Accepted 25 June 2012
Published ahead of print 3 July 2012
Address correspondence to Yuying Lian, email@example.com.
*Present address: Shamika Danzy, Department of Microbiology and Immunology,
Emory University. Atlanta, Georgia, USA, and Naveen Kumar, Division of Animal
Health, Central Institute of Research on Goat, Indian Council of Agricultural
Research, Makhdoom, Mathura, India.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org Journal of Virologyp. 9794–9801September 2012 Volume 86 Number 18
MATERIALS AND METHODS
Cells and viruses. BSRT7-5 cells, which stably express the T7 RNA poly-
merase, were obtained from K. Conzelmann (Ludwig-Maximilians-Uni-
versität, Munich, Germany) and cultured in minimal essential medium
supplemented with 10% fetal bovine serum (FBS), 1 ?g of Geneticin per
ml, and 50 ?g of penicillin-streptomycin/ml. Baby hamster kidney cells
BHK21 and Vero cells were maintained in Dulbecco modified Eagle me-
dium (DMEM) supplemented with 10% FBS and 50 ?g of penicillin-
streptomycin/ml. Human kidney epithelial 293T cells were grown in
DMEM supplemented with 10% FBS and 50 ?g of penicillin-streptomy-
cin/ml. Recombinant PICV viruses were amplified in BHK21 cells, and
the infectious virus titer was determined by plaque assay in Vero cells as
described previously (13).
plasmid DNA by QuikChange PCR-mediated mutagenesis according to
the DNA fragments containing the desired mutation in the PICV Z gene
were subcloned into a plasmid encoding the full-length L segment (14).
To generate PICV Z protein expression constructs, wild-type (WT) or
mutant PICV gene was amplified from the respective plasmid of the full-
length L segment using primers containing hemagglutinin (HA) or Myc
tag at the C terminus, and cloned into the pCAGGS expression vector.
The C-terminal HA-tagged LASV Z was also similarly cloned into the
pCAGGS expression vector. Mutations were introduced into the
Primary sequences of all primers can be provided upon request.
Generation of infectious PICV viruses from plasmid transfection.
Recombinant PICV viruses were generated using the reverse genetics sys-
tem as described previously (14). The L-segment-encoding plasmid of
either WT or mutant Z gene was transfected, along with the S-segment-
encoding plasmid into BSRT7-5 cells. At various time points, superna-
tants were collected for plaque assay in order to quantify the amount of
were verified by sequencing the reverse transcription-PCR (RT-PCR)
products. All rescued mutant viruses have been sequence confirmed.
Growth curve analysis. Cells were seeded in six-well plates at 90 to
of infection (MOI) of 0.01 for 1 h at 37°C. After a washing step with
phosphate-buffered saline, a fresh aliquot of medium was added to the
were harvested for plaque assaying on Vero cells.
PICV Z plasmid for 48 h. Cell pellets were collected and lysed by radio-
immunoprecipitation assay (RIPA) buffer to prepare cell lysates. The Z
self-budding particles were concentrated from the supernatants by poly-
ethylene glycol (PEG) precipitation. In brief, supernatants were centri-
fuged at 14,000 rpm for 10 min. The budded VLPs in the cleared super-
natants were precipitated with 8% PEG 8000–0.5 M sodium chloride,
collected after centrifugation at 14,000 rpm for 15 min, and lysed by de-
and detected for Z proteins by Western blotting with the anti-HA anti-
NP incorporation into Z-induced particles. 293T cells were trans-
or mutant HA-tagged Z expression vector. After 48 h, cell lysates and the
budded VLPs were prepared as described above in the Z budding assay,
for NP and Z using anti-myc and anti-HA antibodies, respectively.
Rescue of recombinant PICV Z mutant viruses using a reverse
in GenBank has revealed multiple conserved residues, including
the G2 myristylation site, the invariant CHCC and CCCC zinc-
binding residues within the central RING domain, the conserved
LP(TK) motif, and the C-terminal proline-rich late (L) domains
(Fig. 1). Previous mutational studies have revealed important
budding assay (18, 19, 23–25) and for the RING domain in regu-
lating viral RNA synthesis (5). Capul et al. have examined the
functional roles of the conserved residues in VLP formation and
infectivity (3). However, their biological roles in the context of
infectious arenavirus replication have never been examined. We
have developed a reverse genetics system for PICV (14), a non-
pathogenic arenavirus whose infection of guinea pigs serves as a
convenient small-animal model for arenavirus-induced hemor-
rhagic fever diseases (1, 11, 27, 28). Except for some sequence
variations in the C-terminal L domains, both PICV and LASV Z
proteins share the same invariant G2, the zinc-binding C/H resi-
dues within the RING domain, and the LPTK residues (Fig. 1).
LASV Z contains two L domains at the C terminus, PTAPP and
PPPYSP, separated by eight residues, whereas PICV Z protein
overlapping L domains PSAPP and PYEP.
We introduced alanine substitution at each of the respective
conserved residues of the PICV Z protein in the L segment of the
reverse genetic system (Table 1). The mutations consist of one to
four amino acid substitutions targeting the G2 myristylation site
(G2A), the central RING domain (C38A/C41A, C51A/H54A/
C57A/C50A, and C71A), the LPTK site [LPTK(78-81)AAAA and
individual mutations], and the L domains (P88A/S89A, P90A/
P91A, and E94A/P95A). To generate recombinant viruses, plas-
mids encoding L and S segments were transfected into cells that
constitutively express the T7 polymerase. At various time points
posttransfection (48, 72, 96, and 120 h), supernatants were col-
lected for plaque assay to determine the amount of infectious
virus particles generated (Table 1). In the WT control, infec-
tious recombinant viruses were produced as early as 48 h post-
transfection (hpt) and reached a titer of ?107PFU/ml at 120
hpt. The successful rate of rescuing WT viruses from this PICV
reverse genetics system is 100%, based on our more than a
hundred experiences thus far. In contrast, despite multiple at-
tempts, most Z mutants were unable to generate any infectious
virus even after 120 hpt, suggesting an essential role for these
conserved residues in the arenaviral life cycle. These lethal mu-
tations include G2A (M32), mutations at the C or H of the
RING domain (M24, M26, and M28), and those of the invari-
at the less conserved residues T80 (M918) and K81 (M40) pro-
duced viable viruses at lowered titers. Dicodon mutations tar-
geting proline residues of the L domains (PSAPPYEP) led to
significantly delayed and reduced production of recombinant
viruses. Compared to WT, both P88A/S89A (M34) and P90A/
P91A (M36) generated ?3 log fewer viruses, whereas E94A/
P95A (M38) produced ? 4 log fewer viruses at 120 hpt (Table
1). Sequence analysis of the RT-PCR products of these mutant
viruses were carried out to verify that no WT revertant viruses
were recovered. In short, we have shown that most of the in-
variant residues of the Z protein play indispensable roles in
arenavirus biology, such as the G2 myristylation site, the C/H
residues within the RING domain, and the LP residues, and
that the proline residues within the L domain play important
and partially overlapping roles in virus replication.
Z Protein in Arenavirus Replication
September 2012 Volume 86 Number 18jvi.asm.org 9795
In vitro growth kinetic analysis of PICV Z mutants. We
plaque purified the five viable recombinant Z mutants—M34
(P88A/S89A), M36 (P90A/P91A), M38 (E94A/P95A), M918
sequence analysis. Compared to WT, K81A produced much
smaller plaques, whereas all four of the other mutants produced
pinhead-sized plaques (Fig. 2A), suggesting that these mutants
have severe growth defects. To quantitatively compare their
T80A grew less well than WT by 0.5 and 1 log, respectively. All
three L domain mutants (P88A/S89A, P90A/P91A, and E94A/
K81 residues reduced viral growth, and those in the L domain
caused even greater growth defects.
Effect of mutations on the Z self-budding activity. A major
known function of the Z protein is to drive the virus budding
process at the cellular membrane (18). We therefore examined
whether any of the Z mutations affect budding. To do this, we
proteins. After plasmid transfection, cell lysates were prepared
and analyzed by Western blotting. WT and all mutant Z proteins
were readily expressed, albeit with some variations at the protein
levels as detected by anti-HA antibody (Fig. 3A, top panels). The
were analyzed by Western blotting with the anti-HA antibody in
residues within the central RING domain are indicated by asterisks (*). The conserved LP(TK) residues and the C-terminal late domains are boxed. (B) The
three-dimensional model of LASV Z protein is shown in tube form (magenta, PDB accession no. 2KO5), with the G2 residue, LPTK motif, and the two late
domains (PTAPP and PPPYSP) shown in yellow. The central RING domain binds two zinc cations that are represented by gray balls.
Wang et al.
jvi.asm.org Journal of Virology
both reducing (VLPs) and nonreducing (VLPs, no dithiothreitol
[?DTT]) conditions in order to detect Z monomeric and multi-
meric formations (Fig. 3A, bottom panel). The relative budding
to those in the cells, followed by a comparison of mutant to WT
(set at 1.0). Consistent with a previous report (19), we found that
lane 2), whereas mutation of the G2 myristylation residue (G2A)
completely abrogated self-budding activity (lane 3). The three
completely lost (lanes 5 and 6) or significantly reduced the self-
ity of the G2A and RING domain mutations likely explains their
inability to support fully infectious viruses (Table 1). In contrast,
dicodon mutations within the C-terminal L domain of PICV
(P88A/S89A, P90A/P91A, and E94A/P95A) resulted in little to no
decrease in budding activity (relative budding efficiency varies
from 0.5 to 0.9) (Fig. 3A, lanes 8 to 10), which correlates with the
phenotypes of these L domain mutants in viral viability and
mutations of the LPTK residues had little effect on Z budding
the two invariant residues have essential functions other than vi-
similar alanine substitutions were introduced into the HA-tagged
LASV Z protein expression vector. These mutations target the G2
site (G2A), the RING domain (C31A/C34A, CHCCCC, and
proteins were efficiently expressed, albeit with some variations
both reducing (VLPs) and nonreducing (VLPs, ?DTT) condi-
tions (Fig. 3B, bottom panel). Most mutations including those at
the LPTK site and the L domains had little effects on Z budding
budding and a major CHCCCC mutation in the RING domain
that significantly reduced budding efficiency. The LPTK¡AAAA
mutation appeared to decrease the level of Z monomers and
but nonetheless did not affect the overall relative budding effi-
sion level in the cytoplasm.
Overall, mutational analyses of the conserved residues of both
PICV and LASV Z proteins have produced similar results on the
self-budding activity with some variations observed for the RING
domain mutants. The RING domain of PICV Z protein seems to
play an important role in Z budding, as three RING domain mu-
tants had little or no budding activity (Fig. 3A, lanes 4 to 6). In
contrast, the RING domain of LASV Z protein seems to play a
less important role in Z budding, since only a 6-residue change
(CHCCCC) could lead to a notable decrease in budding (Fig. 3B,
lane 4), while two other dicodon mutations (C31A/C34A and
FIG 2 Rescue of recombinant PICV viruses with wild-type or mutant Z pro-
teins. (A) Plaque morphology of viable recombinant PICV viruses encoding
WT or the respective Z mutant proteins. (B) Determination of viral growth
kinetics of recombinant viruses by growth curve analysis. Vero cells were in-
in the supernatants at various time points postinfection were quantified by
plaque assay. The results shown are averages and standard deviations from at
least three independent experiments.
TABLE 1 Infectious titers of the recombinant PICV Z mutants
generated by reverse genetics systema
mutant Z mutation
Infectious titers (PFU/ml)
48 hpt72 hpt 96 hpt120 hpt
4.8E ? 04
7.8E ? 05
4.2E ? 04
1.2E ? 07
2.4E ? 04
3.6E ? 05
aRecombinant PICV strains were generated by the reversegenetics systems (14), in
which a plasmid encoding thefull-length L segment of either the WT or the respective
mutant Z gene, alongwith a plasmid encoding the S segment, were transfected into
BSRT7-5cells. Supernatants collected at 48, 72, 96, and 120 h posttransfection
(hpt)were subjected to plaque assay to determine the titer of infectious virus. Theresults
shown are representative of three independent experiments.
Z Protein in Arenavirus Replication
September 2012 Volume 86 Number 18jvi.asm.org 9797
PICV and LASV Z proteins, the G2 myristylation site plays an
essential role in Z budding activity, the C-terminal L domains
have redundant functions, and the LP(TK) site is not at all re-
quired in this process.
Effect of mutations on NP incorporation into VLPs. It has
been demonstrated that NP can be incorporated into the Z-in-
duced VLPs possibly through specific NP-Z interactions (3, 4).
We examined whether Z mutations affect NP incorporation into
VLPs by cotransfecting 293T cells with LASV NP-myc expression
vector together with either WT or mutant Z-HA vectors. Similar
results were observed for PICV Z mutants (data not shown). Cell
lysates and VLPs in the supernatants were analyzed for NP and Z
by Western blotting with anti-myc and anti-HA antibodies, re-
spectively (Fig. 4). The relative Z budding efficiency was deter-
mined as described above, and the relative NP incorporation effi-
ciency was calculated by the level of NP expression normalized to
precipitates from cells expressing NP alone, suggesting that, in
contrast to Z, NP does not have a self-budding activity (Fig. 4,
lanes 1 to 2). However, NP was readily detected in VLPs when the
NP can be efficiently incorporated into Z-induced VLPs. Com-
pared to the Z budding activity in the absence of NP as shown in
Fig. 3B, the presence of NP did not significantly alter the budding
mutants (C31A/C34A and CHCCCC) and one L domain mutant
cies of 0.5, 0.2, and 0.2, respectively) (Fig. 4, lanes 5, 6, and 9); the
cells were transfected with either WT or the respective PICV Z mutant expression vectors. Expressions of HA-tagged Z proteins (Z-HA), along with ?-actin
was conducted on the WT and mutant LASV Z proteins.
FIG 4 Effects of mutations at the conserved residues of Z on the efficiency of
with WT or mutant Z protein expression vector and with or without the NP
expression vector. The amounts of VLPs released into the supernatants were
analyzed by Western blot against Z and NP proteins. The relative Z budding
efficiency was quantified as described in the legend of Fig. 3. The relative NP
incorporation efficiency for each Z mutant was determined by the relative NP
levels incorporated into the released VLPs compared to the WT Z control (set
Wang et al.
jvi.asm.orgJournal of Virology
reason for this is unclear. Since G2A mutant did not form VLPs,
other hand, NP was present at various levels in the VLPs induced
by each of the other Z mutants (Fig. 4, lanes 5 to 14), indicating
Z mutants with reduced relative NP incorporation efficiency. For
example, the RING domain mutant C64A/C67A significantly de-
The LPTK¡AAAA mutation and three individual mutations
(L71A, P72A, and T73A) all reduced NP incorporation by 50 to
60% (Fig. 4, lanes 8 and lanes 11 to 13), while the K74A mutation
reduced it by 30% (Fig. 4, lane 14). In contrast, the CHCCCC
RING domain mutant and the PPPY late domain mutant in-
(Fig. 4, lanes 6 and 9), as a result of their substantially lowered
levels of Z budding but nonetheless normal amounts of incorpo-
rated NPs. In short, we have shown that none of the conserved
residues in the Z protein is essential for incorporating NP into
play a more important role in this process.
Effect of mutations on inhibiting viral RNA synthesis in
minigenome assay. Arenavirus Z protein has been shown to reg-
ulate viral RNA replication and transcription by inhibiting viral
RNA synthesis (6). We ask whether any of our Z mutants could
affect viral RNA synthesis by using our recently established mini-
genome system (20). This system consists of the LASV S segment
that is devoid of viral gene coding sequences but instead encodes
the Renilla luciferase reporter (RLuc) gene in a negative-sense
well as the intergenic region. This 5RLuc3 minigenome is recog-
nized by LASV or PICV polymerase complex that consists of the
respective NP and L proteins and can produce up to 5- to 6-log-
higher levels of RLuc expression than a control reaction that lacks
the L-protein-encoding plasmid (Fig. 5, control versus no Z). As
RNA synthesis in both LASV and PICV minigenomic systems
LASV and PICV Z proteins, all RING domain mutations and the
inhibit viral RNA synthesis, whereas all L domain mutants func-
tioned just like the WT (Fig. 5). Interestingly, whereas the G2A
mutant of LASV Z protein still strongly inhibited viral RNA syn-
thesis at the same level as the WT, the same mutation in the PICV
Z context completely lost the inhibitory activity (Fig. 5, shown by
arrows). In summary, we found that the RING domain and the
LPTK residues are essential in regulating viral RNA synthesis and
that the G2 residue in different Z proteins may have a differential
the invariant residues of arenaviral Z protein in infectious viral
replication. These residues, including G2, the zinc-binding C and
known Z proteins. Previous studies have demonstrated some of
these residues’ critical roles in VLP formation and viral RNA reg-
ulation (3, 5, 19, 24). Our study, however, establishes their essen-
tial roles in the context of infectious virus life cycle, since alanine
substitutions at these residues have resulted in nonproductive
PICV virus production (Table 1). The proline-rich L domain can
tolerate mutations to some levels; however, these mutations have
dramatically altered viral plaque formation and significantly re-
duced viral growth ability, suggesting an important role of the
intact L domain in viral replication. Our comprehensive analyses
of key residues of arenaviral Z proteins have also provided some
novel insights into their mechanistic roles in virus budding, nu-
cleocapsid incorporation, and viral RNA regulation, as discussed
An essential biological role of the Z protein in arenavirus in-
lications (19, 24) and the present study (Fig. 3), the G2 myristyla-
tion site is indispensable for the Z protein-mediated budding
activity, which most likely explains the vital role of G2 in arenavi-
rus infection (Table 1). Also important in mediating Z budding is
FIG 5 Effects of mutations at the conserved residues on Z-mediated inhibition of viral RNA synthesis. LASV Z (left panel) or PICV Z (right panel) proteins of
either wild-type or mutant forms were examined for their ability to inhibit viral RNA transcription in the minigenome assay. Arrows show differential effects of
G2A mutation between LASV and PICV Z proteins. The results shown are averages of at least three independent experiments with error bars representing
Z Protein in Arenavirus Replication
September 2012 Volume 86 Number 18jvi.asm.org 9799
its C-terminal L domain(s) (18, 23, 25). The L domain consists of
ponents of the ESCRT pathway to mediate membrane budding.
Thus far, three classes of viral L domains have been defined, PT/
SAP, LxxLF or YPXL, and PPxY, which interact with Tsg101,
ALIX, and NEdd4-like HECT ubiquitin ligases, respectively (10).
These L domains are functionally interchangeable (26). Some
arenaviral Z proteins, such as LASV Z, contain two separate L
domains PTAPP and PPPYSP, whereas others contain a proline-
rich region that seems to consist of two overlapping L domains
(e.g., PSAPPYEP for PICV) or just one L domain (e.g., PTAPPP
for Junin) (Fig. 1). Although dicodon mutation of the Proline
residues in PSAPPYEP of PICV Z protein has minor effect on its
self-budding activity (Fig. 3A, lanes 8 to 10), possibly due to the
intact L domain(s) of arenaviral Z proteins are required for effi-
cient virus budding and replication.
into arenavirus virions is largely unknown. It has been proposed
that the interaction between Z and NP is required to recruit and
incorporate the nucleocapsids into budding virions at the cell
a single residue/domain of Z that is indispensable for NP incor-
RING domain, may play a more important role.
The central RING domain is a zinc-binding motif with invari-
shown that the zinc-binding residues are absolutely essential for
arenavirus replication (Table 1), the functional role(s) of the
RING domain in arenavirus biology is less clear. It seems unlikely
that the lethal phenotype observed for all RING domain muta-
tions is due to the abolishment of either Z budding or NP incor-
abolish the Z protein’s ability to inhibit viral RNA synthesis (Fig.
esis is that this inhibition is necessary in order to initiate the viral
biological significance of Z-mediated viral RNA inhibition in
arenavirus life cycle.
We have demonstrated an essential role for L78 or P79 (num-
Using the PICV reverse genetics system, we have shown that both
of viral infectivity (Table 1). This is consistent with a recent study
LASV Z protein strongly reduce VLP infectivity. Their functional
We and other researchers have shown that mutagenesis of the
4; the present study). Furthermore, both lethal (L78A and P80A)
an ?50% decrease in NP incorporation. Therefore, we do not
believe that the indispensable role of L and P residues in the viral
life cycle is to mediate the NP incorporation into viral particles.
that both L¡A and P¡A mutations abolish Z-mediated inhibi-
tion of viral RNA synthesis, which may explain their inability to
support infectious viral replication as discussed above for the
RING domain mutants. Nevertheless, the exact functional mech-
anisms of the invariant L and P residues in arenaviral infectious
life cycle remain to be fully elucidated.
of both LASV and PICV Z protein in budding (Fig. 3) and RNA
regulation (Fig. 5). Most of the conserved residues/domains have
RNA inhibition. There are, however, some interesting variations.
A notable one is the role of G2 in blocking viral RNA synthesis
(Fig. 5). The G2 residue is indispensable for PICV Z-mediated
viral RNA inhibition but is unnecessary for LASV Z. A previous
study has shown that the Z protein of LCMV, another Old World
arenavirus, does not require the N-terminal residues 1 to 16 (in-
cluding G2) to inhibit viral RNA synthesis (5). It remains to be
determined whether the G2 residue of other arenavirus proteins
plays an important role in viral RNA regulation. In addition, a
recent study using Machupo virus polymerase complex in an in
vitro polymerase assay has suggested that Z directly interacts with
the L polymerase protein to block the early steps of viral RNA
synthesis in a species-specific manner (12). Therefore, the func-
tional mechanism of Z in viral RNA regulation requires further
characterization in specific arenavirus species.
tial roles of the conserved residues (domains) of the Z protein in
the infectious arenavirus life cycle. In addition, our studies have
studies may lead to the development of novel antivirals targeting
the essential Z protein in order to treat arenavirus-induced dis-
We thank K. Conzelmann (Ludwig-Maximilians-Universität) for the
This study was supported in part by NIH grants R01AI083409 to Y.L.
and R56AI091805 and R01AI093580 to H.L.
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features of arenavirus disease in guinea pigs: comparison of two Pichinde
virus strains. Am. J. Pathol. 145:228–235.
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