JOURNAL OF VIROLOGY, Dec. 2011, p. 13038–13048
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 24
The C-Terminal Region of Lymphocytic Choriomeningitis Virus
Nucleoprotein Contains Distinct and Segregable Functional
Domains Involved in NP-Z Interaction and Counteraction
of the Type I Interferon Response?
Emilio Ortiz-Rian ˜o,1Benson Yee Hin Cheng,1Juan Carlos de la Torre,2* and Luis Martínez-Sobrido1*
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642,1and
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 920372
Received 28 July 2011/Accepted 21 September 2011
Several arenaviruses cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity
and significant mortality. Arenavirus nucleoprotein (NP), the most abundant viral protein in infected cells and
virions, encapsidates the viral genome RNA, and this NP-RNA complex, together with the viral L polymerase,
forms the viral ribonucleoprotein (vRNP) that directs viral RNA replication and gene transcription. Formation
of infectious arenavirus progeny requires packaging of vRNPs into budding particles, a process in which
arenavirus matrix-like protein (Z) plays a central role. In the present study, we have characterized the NP-Z
interaction for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). The LCMV NP domain
that interacted with Z overlapped with a previously documented C-terminal domain that counteracts the host
type I interferon (IFN) response. However, we found that single amino acid mutations that affect the anti-IFN
function of LCMV NP did not disrupt the NP-Z interaction, suggesting that within the C-terminal region of NP
different amino acid residues critically contribute to these two distinct and segregable NP functions. A similar
NP-Z interaction was confirmed for the HF arenavirus Lassa virus (LASV). Notably, LCMV NP interacted
similarly with both LCMV Z and LASV Z, while LASV NP interacted only with LASV Z. Our results also
suggest the presence of a conserved protein domain within NP but with specific amino acid residues playing
key roles in determining the specificity of NP-Z interaction that may influence the viability of reassortant
arenaviruses. In addition, this NP-Z interaction represents a potential target for the development of antiviral
drugs to combat human-pathogenic arenaviruses.
Arenaviruses cause chronic infections of rodents with a
worldwide distribution (8). Humans become infected through
mucosal exposure to aerosols or by direct contact of skin abra-
sions with infectious material. Several arenaviruses cause hem-
orrhagic fever (HF) disease in humans and pose a serious
public health problem in their regions of endemicity (8, 41, 52).
Moreover, increased travel to and from regions of endemicity
has resulted in importation of HF cases into metropolitan
areas of regions of nonendemicity (28). On the basis of their
antigenic features and phylogenetic relationships, arenaviruses
are classified into Old World arenaviruses (OWAs) and New
World arenaviruses (NWAs) (8). Due to its vast region of
endemicity and the size of the population at risk, the OWA
Lassa virus (LASV), the causative agent of Lassa fever (LF), is
the HF arenavirus with the highest impact on public health (21,
26). Nevertheless, several NWAs, especially Junin virus
(JUNV), the causative agent of Argentine HF (AHF) (64), are
also clinically relevant human pathogens (23). In addition,
evidence indicates that the globally distributed prototypic are-
navirus lymphocytic choriomeningitis virus (LCMV) is likely a
neglected human pathogen (30) of clinical significance in con-
genital infections (1, 44). Moreover, LCMV infections of im-
munocompromised individuals can result in severe disease and
death (17, 48). The potential for newly emerging highly patho-
genic arenaviruses is also worth noting, as has been illustrated
by the recent isolation of Lujo virus from patients with HF
disease in South Africa (7). In addition, several arenaviruses
have been included as category A agents because they could
potentially be used as agents of bioterrorism (4, 10). Public
health concerns posed by human-pathogenic arenaviruses are
aggravated by the lack of Food and Drug Administration
(FDA)-licensed vaccines and because current antiarenaviral
therapy is limited to off-label use of the nucleoside analog
ribavirin, which is only partially effective (31, 42, 43). More-
over, efficient ribavirin therapy requires early and intravenous
administration and is often associated with significant side ef-
fects (56, 60). All these reasons underscore the importance of
developing novel antiviral strategies to combat arenavirus in-
fections, a task that would be facilitated by a better under-
standing of the molecular and cell biology of arenaviruses.
Arenaviruses are enveloped viruses with a bisegmented neg-
ative-strand RNA genome. Each genome segment, designated
L (ca. 7.3 kb) and S (ca. 3.5 kb), encodes two viral proteins
using an ambisense coding strategy (8). The L RNA encodes
the viral RNA-dependent RNA polymerase (L) and the small
* Corresponding author. Mailing address for Luis Martínez-So-
brido: Department of Microbiology and Immunology, University of
Rochester School of Medicine and Dentistry, 601 Elmwood Avenue,
Rochester, NY 14642. Phone: (585) 276-4733. Fax: (585) 473-9573.
E-mail: firstname.lastname@example.org. Mailing address for Juan
C. de la Torre: Department of Immunology and Microbial Science,
The Scripps Research Institute, La Jolla, CA 92037. Phone: (858)
784-9462. Fax: (858) 784-9981. E-mail: email@example.com.
?Published ahead of print on 5 October 2011.
RING finger protein called Z, which has been shown to be the
arenavirus counterpart of the matrix (M) protein found in
many negative-strand RNA viruses. As with many M proteins,
arenavirus Z plays a critical role in virion assembly and is the
major driving force of arenavirus budding (15, 50, 51, 61, 63).
Z has also been shown to regulate viral transcription and
replication (12–14, 18, 35) and to interact with the virus poly-
merase L (29, 65) and a variety of cellular proteins, including
the eukaryotic translation initiation factor 4E, the promyelo-
cytic leukemia protein (PML), the ribosomal P0 protein (3, 9),
and the intracellular sensor retinoic acid-inducible gene I
(RIG-I) (16). The S RNA encodes the viral glycoprotein pre-
cursor (GPC) and the nucleoprotein (NP). GPC is posttrans-
lationally processed by the cellular protease S1P to produce
GP-1 and GP-2 (6, 8), which forms the glycoprotein complex
GP that makes up the spikes that decorate the surface of the
virion structure and mediate receptor recognition and cell en-
try (8). NP is the most prevalent viral protein in infected cells
and virions. NP encapsidates the viral genome, and this NP-
RNA complex, together with L, forms the viral ribonucleopro-
tein (vRNP) particle that is the functional unit for both RNA
replication and gene transcription of the viral genome in the
cytoplasm of infected cells (32, 33, 53). Besides its critical role
in replication and transcription, NP counteracts the host type I
interferon (IFN) response during viral infection by preventing
activation and nuclear translocation of the interferon regula-
tory factor 3 (IRF-3) and subsequent induction of IFN pro-
duction and interferon-stimulated genes (ISGs) (5, 40). This
anti-IFN function of NP is shared by all members of the family
examined, with the exception of Tacaribe virus (TCRV) NP
(39). Mutation-function studies mapped this anti-IFN function
to the C-terminal region of NP, which contains a functional
3?-5? exonuclease domain (25, 54) whose activity was linked to
the anti-IFN activity of NP (38).
For many enveloped viruses, the interaction of vRNPs with
viral matrix proteins has been shown to be required for for-
mation of mature infectious progeny (24, 57, 58), and NP-Z
interaction has been suggested to mediate the incorporation of
vRNPs into matured infectious virions (20). Accordingly, re-
cent findings have shown that the C-terminal region of arena-
virus NPs interacts with their respective Z (matrix) proteins
(34, 59). However, the mechanisms by which arenavirus vRNPs
are recruited by Z into mature enveloped infectious progeny
are little understood. Likewise, it remains unknown whether
NP domains responsible for NP-Z interaction are also involved
in other NP functions. Moreover, the role of heterotypic NP-Z
interactions in the generation of novel viable reassortant are-
naviruses has not been investigated.
In this work, we provide experimental evidence that LCMV
NP interacts with LCMV Z and that this interaction is medi-
ated by a C-terminal region of NP that overlaps with the
previously described NP domain involved in counteracting the
host IFN response (38). However, specific single amino acid
mutations that impaired NP anti-IFN function did not affect
NP’s ability to interact with Z, suggesting that different resi-
dues within the C-terminal region of NP critically contribute to
these two distinct NP activities. Notably, we observed that
LCMV NP also interacted efficiently with LASV Z, whereas
LASV NP interacted with LASV Z but not with LCMV Z. This
finding suggests that restrictions in the directionality of NP-Z
interaction may influence the viability of reassortant arenavi-
ruses. Furthermore, the observation that NPs lacking the N-
terminal region involved in NP self-association (47) were still
able to interact with Z suggests that monomeric NP can both
interact with Z and inhibit the cellular type I IFN response.
The identification of this NP functional domain involved in
NP-Z interaction would facilitate further studies aimed at a
detailed understanding of arenavirus assembly and production
of infectious progeny. Likewise, this NP-Z interaction repre-
sents a novel target for the development of antiviral drugs
capable of disrupting NP-Z interaction and thereby inhibiting
production of infectious progeny.
MATERIALS AND METHODS
Cells and viruses. Baby hamster kidney (BHK-21) cells (ATCC CCL-10),
human embryonic kidney (293T) cells (ATCC CRL-11268), Madin-Darby canine
kidney (MDCK) cells (ATCC CCL-34), and African green monkey kidney epi-
thelial (Vero) cells (ATCC CCL-81) were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, L-glu-
tamine (2 mM), penicillin (100 units/ml), and streptomycin (100 ?g/ml). Cells
were grown at 37°C in a 5% CO2atmosphere. For viral infections, cells were
maintained in a mixture (1:1) of Opti-MEM reduced serum medium and the
normal medium for each cell type. LCMV (Armstrong strain ARM53b) was
produced by infecting BHK-21 cells (multiplicity of infection [MOI] ? 0.1) and
harvesting the supernatants on day 3 or 4 postinfection (55). LCMV titers were
determined by immunofocus centers on Vero cells as described previously (2).
Plasmids. Hemagglutinin (HA)- and FLAG-tagged versions of NP and Z of
LCMV and LASV were obtained by cloning the corresponding open reading
frames (ORFs) into modified versions of the pCAGGs multicloning site (MCS)
plasmid (46). To that end, the pCAGGs MCS plasmid was modified by cloning
the HA (YPYDVPDYA) and FLAG (DYKDDDDK) epitopes, allowing the
generation of N- or C-terminus-tagged versions of proteins as described previ-
For the mammalian two-hybrid (M2H) system, the herpes simplex virus VP16
transactivating domain and the GAL4 DNA-binding domain ORFs were ampli-
fied by PCR and cloned into the pCAGGs MCS plasmid to generate pCAGGs
VP16 and pCAGGs GAL4 plasmids, respectively, containing two flanking MCSs
to facilitate the generation of N-terminal and C-terminal fusion proteins. HA-
tagged versions of LCMV and LASV NP and Z ORFs were amplified by PCR
and cloned into pCAGGs VP16 and pCAGGs GAL4 plasmids. Previously de-
scribed N-terminal and C-terminal deletion mutants, as well as single amino acid
substitutions of LCMV NP to alanine (38), were subcloned into the pCAGGs
VP16 plasmid to generate NP-VP16 fusion proteins. Amino acids participating in
the active site of the 3?-5? exonuclease domain located within the C terminus of
LCMV NP (25, 54) were replaced by alanine (D382A, E384A, D459A, H519A,
and D522A) by site-directed mutagenesis (Stratagene), using a pGEM-T LCMV
NP template, and subcloned into the pCAGGs VP16 plasmid. The pG5Luc
reporter plasmid (Promega) was modified by fusing the green fluorescent protein
(GFP) ORF to the N-terminal region of the firefly luciferase (FFL) coding
sequence to generate pG5 GFP/Luc.
For the bimolecular fluorescence complementation (BiFC) system, N-terminal
(EYN) and C-terminal (EYC) sequences of the yellow fluorescent protein (YFP)
ORF were amplified by PCR from pCAGGs EYN-NS1 and pCAGGs EYC-NS1
and cloned into the pCAGGs MCS vector to generate pCAGGs EYN and
pCAGGs EYC, respectively, containing two flanking MCSs to facilitate the
generation of N- and C-terminal fusion proteins. LCMV NP and Z were sub-
cloned into EYN and EYC pCAGGs to generate N- and C-terminal fusion
Primers for the generation of the described plasmids are available upon re-
quest. The coding regions of the generated constructs were verified by DNA
sequencing, and protein expression was verified by Western blotting.
M2H assay. 293T cells (6.5 ? 105per transfection) were cotransfected in
suspension with 2 ?g of the indicated pCAGGs VP16 and GAL4 expression
plasmids, 1 ?g of the reporter pG5 GFP/Luc plasmid, and 0.1 ?g of the simian
virus 40 (SV40)-Renilla luciferase expression vector pRL SV40 (Promega), to
normalize transfection efficiencies, using 1 ?g of Lipofectamine 2000 per ?g of
plasmid DNA. Transfected cells were seeded onto 12-well tissue culture plates.
At 48 h posttransfection, protein-protein interaction was evaluated by GFP
expression using a Zeiss fluorescence microscope. After imaging, cell lysates
VOL. 85, 2011 LCMV NP DOMAIN INVOLVED IN NP-Z INTERACTION13039
were prepared to determine luciferase activities and protein expression. Lucif-
erase activities were determined using the dual-luciferase reporter assay (Pro-
mega) and a Lumicount luminometer (Packard). Reporter gene activation is
expressed as fold induction over the negative controls (pCAGGs NP-VP16- and
pCAGGs GAL4-transfected cells). The percentage of interaction of LCMV NP
mutants was calculated on the basis of the wild-type NP-Z interaction. All M2H
experiments were performed in triplicate. The mean and standard deviation were
calculated using Microsoft Excel software. Protein expression was determined by
Western blotting using the indicated antibodies.
BiFC assay. MDCK cells (105) were transfected in suspension with 1 ?g of the
indicated pCAGGs EYN and EYC plasmids using 1 ?g of Lipofectamine 2000
per ?g of plasmid DNA and seeded onto coverslips placed on 24-well tissue
culture plates. At 24 h posttransfection, cells were cultured for 3 h at 30°C in a
5% CO2atmosphere to allow maturation of the YFP (27). Cells were then fixed
with 100% methanol for 5 min, permeabilized with 0.1% Triton X-100 for 10
min, and blocked in 2.5% bovine serum albumin (BSA) in 1? phosphate-
buffered saline (1? PBS) for 1 h at room temperature. Samples were incubated
for 1 h at 37°C with a 1:500 dilution in 2.5% BSA of a monoclonal antibody
against GFP (AB1218; AbCAM) that recognizes only the reconstituted form of
the fluorescent protein. Subsequently, cells were washed with 1? PBS and
incubated with 4?,6-diamidino-2-phenylindole (DAPI; Research Organics) and a
1:1,000 dilution of secondary goat anti-mouse immunoglobulin G (IgG)-Alexa
Fluor 647 (Invitrogen) for 30 min at 37°C. Cells were washed with 1? PBS, and
coverslips were mounted with Mowiol solution onto glass slides and analyzed
using a 63? oil immersion objective and a Zeiss fluorescence microscope. Images
were colored using Adobe Photoshop CS4 (version 11.0) software. Representa-
tive images of at least three independent transfections are shown.
Immunofluorescences. Vero cells (105per transfection) on coverslips were
cotransfected with 1.5 ?g of the indicated LCMV and LASV NP and Z expres-
sion plasmids using 1 ?g of Lipofectamine 2000 per ?g of DNA. Empty pCAGGs
MCS plasmid was included to maintain a constant amount of transfected plasmid
DNA. At 48 h posttransfection, cells were fixed with 4% formaldehyde for 15 min
at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at
room temperature, followed by an overnight blocking step with 2.5% BSA in 1?
PBS. After blocking, cells were incubated for 1 h at 37°C with the indicated
primary antibodies: an anti-HA mouse monoclonal antibody (1:500; H9658;
Sigma) and an anti-FLAG rabbit polyclonal serum (1:500; F7425; Sigma). After
incubation, cells were washed with 1? PBS and incubated with DAPI (Research
Organics), a 1:500 dilution of goat anti-mouse IgG-Alexa Fluor 488 (for the
monoclonal antibody), and a 1:300 dilution of goat anti-rabbit IgG-rhodamine
red secondary antibodies for 30 min at 37°C. Cells were then washed with 1?
PBS, and coverslips were mounted with Mowiol solution onto glass slides and
analyzed by florescence microscopy using a 63? oil immersion objective. For
NP-Z colocalization during LCMV infection, subconfluent monolayers of Vero
cells on glass coverslips were infected with LCMV (MOI ? 0.1), and at 48
hpi, cells were fixed, permeabilized, and blocked as described above. After
blocking, cells were incubated for 1 h at 37°C with a monoclonal antibody against
LCMV NP (1.1.3) and a polyclonal rabbit serum against LCMV Z. After incu-
bation, cells were washed 3 times with 1? PBS and incubated with DAPI, goat
anti-mouse IgG-Alexa Fluor 488 (for NP monoclonal antibody), and goat anti-
rabbit IgG-rhodamine red (for Z polyclonal antibody). Coverslips were mounted
with Mowiol solution and analyzed as described above. Mock-infected cells were
included as controls. Representative images of three independent transfections
or infections are shown.
Protein gel electrophoresis and Western blotting analysis. Proteins were sep-
arated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-
Rad) overnight at 4°C. After blocking for 1 h at room temperature with 10% dry
milk in 1? PBS, membranes were incubated with anti-HA mouse monoclonal
antibody (1:1,000; H9658; Sigma), anti-FLAG polyclonal antibodies (1:1,000;
F7425; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH)
monoclonal antibody (1:1,000; AB9484; AbCAM), anti-VP16 polyclonal anti-
body (1:5,000; V4388; Sigma), and anti-GFP polyclonal antibody (1:500; Santa
Cruz, SC8334). Incubation with antibodies was done overnight at 4°C. After
overnight incubation, membranes were washed three times with 1? PBS con-
taining 0.1% Tween 20 and incubated with a 1:2,000 dilution of the respective
secondary horseradish peroxidase-conjugated anti-mouse and/or anti-rabbit Ig
antibodies (GE Healthcare United Kingdom) for 1 h at room temperature. After
3 washes with 1? PBS containing 0.1% Tween 20, proteins were detected using
a chemiluminescence kit and autoradiography films from Denville Scientific Inc.
Protein band intensities were quantified using ImageJ software (NIH) and are
represented as a percentage of wild-type NP expression levels.
Generation and isolation of VLPs. To generate virus-like particles (VLPs),
293T cells were cotransfected in suspension (6-well plate format, 106cells/well)
with 2.5 ?g of each indicated plasmid using 1 ?g of Lipofectamine 2000 per ?g
of DNA. Empty pCAGGs MCS plasmid was used to keep a constant amount of
transfected plasmid DNA. At 72 h posttransfection, cells and tissue culture
supernatants were collected. Supernatants were clarified at 10,000 rpm for 30
min and then layered on top of a 20% sucrose cushion and centrifuged at 35,000
rpm on an SW-41 rotor for 2.5 h. VLP-containing pellets were resuspended in
100 ?l of 1? PBS, and 20 ?l was analyzed by Western blotting. Cell pellets were
lysed with 400 ?l of lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 100 mM
NaCl, 1% NP-40, complete cocktail of protease inhibitors; Roche) for 30 min on
ice. Cell lysates were clarified by centrifugation at 14,000 rpm for 30 min at 4°C.
Twenty microliters (5% of total cell lysates) of each sample was analyzed (input)
by Western blotting.
Assessment of LCMV NP-Z interaction. Z is the driving
force of arenavirus budding and in the absence of other viral
proteins mediates formation of VLPs (50, 51, 61, 63). In addi-
tion, the Z proteins of LASV (15), Mopeia virus (MOPV) (59),
FIG. 1. LCMV NP-Z interaction. (A) Schematic representation of
LCMV NP and Z WT proteins fused to VP16 and GAL4, respectively,
used in the M2H assay system to detect NP-Z interaction. LCMV NP
and Z were fused to the N- and C-terminal domains, respectively, of
VP16 (NP-VP16) and GAL4 (GAL4-Z). (B and C) LCMV NP-Z
interaction in the M2H assay. 293T cells (12-well format) were cotrans-
fected with 2 ?g of the NP-VP16 and GAL4-Z pCAGGs expression
plasmids, together with 1 ?g of the dual reporter plasmid pG5 GFP/
Luc (to detect protein-protein interaction) and 0.1 ?g of the SV40-
Renilla luciferase expression vector pRL SV40 (to normalize transfec-
tion efficiencies). GAL4 and VP16 expression plasmids were included
as controls to demonstrate the specificity of the LCMV NP-Z interac-
tion. Forty-eight hours after transfection, GFP expression was assessed
using fluorescence microscopy (B) and cell extracts prepared to deter-
mine the strength of the interaction using a dual-luciferase reporter
assay. Renilla luciferase values (means ? standard deviations) are
indicated in each image. Fold induction over the negative control
(NP-VP16 plus GAL4) is indicated (C). Luciferase activities were
determined using the dual-luciferase reporter assay. Reporter gene
activation is expressed as fold induction over the negative controls
(pCAGGs NP-VP16- and pCAGGs GAL4-transfected cells).
13040 ORTIZ-RIAN ˜O ET AL.J. VIROL.
and TCRV (34) have been shown to mediate incorporation of
NP into VLPs. To characterize the NP-Z interaction for the
prototypic arenavirus LCMV, we used two complementary ap-
proaches: the M2H system and the BiFC assay. For the M2H
system (Fig. 1), we cotransfected 293T cells with pCAGGs
plasmids expressing NP-VP16 and GAL4-Z (Fig. 1A), together
with the reporter plasmid pG5 GFP/Luc and with pRL SV40 to
normalize transfection efficiencies. In this system, the LCMV
NP-Z interaction would allow GAL4 binding to the reporter
plasmid, while the VP16 transactivation domain would recruit
the machinery necessary for reporter gene expression (GFP
fused to FFL). We used pCAGGs VP16 and GAL4 plasmids as
negative controls, alone or in combination with LCMV NP-
and Z-tagged expression plasmids, to demonstrate the speci-
ficity of the LCMV NP-Z interaction. We detected the LCMV
NP-Z interaction using both GFP (Fig. 1B) and FFL (Fig. 1C)
reporter gene expression. Reporter gene expression was not
detected when either GAL4 or VP16 was used alone or in
combination with LCMV NP or Z protein, demonstrating the
specificity of the interaction.
To confirm and further characterize this LCMV NP-Z in-
teraction, we used the BiFC assay (Fig. 2). For this, we fused
the N terminus (residues 1 to 155; EYN) and C terminus
(residues 156 to 239; EYC) of YFP to either the NP or Z
protein from LCMV (Fig. 2A). The NP-Z interaction should
restore the YFP ternary structure and associated fluorescence
properties. We confirmed that all fusion constructs were ex-
pressed, as determined by Western blotting using an anti-GFP
polyclonal antibody that detects the N- and C-terminal do-
mains of both GFP and YFP (Fig. 2B). The differences in the
apparent molecular mass of LCMV Z observed depending on
whether EYN or EYC was fused to the N or C terminus were
likely due to differences in the links between the N- or C-ter-
minal EYN or EYC expression plasmids. In the case of con-
struct Z-EYC, we observed an extra band with faster mobility
that likely reflects a degradation product. We then cotrans-
fected MDCK cells with the indicated plasmid combinations
(Fig. 2C). In cells cotransfected with NP-EYN and EYC-Z and
with NP-EYC and EYN-Z, we observed YFP expression in-
dicative of LCMV NP-Z interaction (Fig. 2C, YFP). To con-
FIG. 2. Detection of LCMV NP-Z interaction using the BiFC assay. (A) Schematic representation of LCMV NP and Z proteins fused to the
N-terminal (EYN) and the C-terminal (EYC) YFP. (B) Protein expression levels. Cell lysates of 293T cells transfected with the indicated plasmids
were prepared and analyzed by Western blotting using an anti-GFP polyclonal antibody (?-GFP) that detects the N- and C-terminal regions of
YFP. GAPDH was used as a loading control. Protein molecular mass markers (kDa) are indicated on the left. (C) Reconstitution of YFP
fluorescence. MDCK cells were cotransfected (24-well plate format) with 1 ?g of the indicated plasmid combinations. At 24 h posttransfection,
cells were incubated at 30°C for 3 h to allow maturation of YFP. Reconstitution of YFP was examined by fluorescence microscopy (YFP) and using
an anti-GFP monoclonal antibody that recognizes only the reconstituted GFP or YFP (?-GFP), and DAPI staining of the cell nuclei and merge
images are shown. Representative images are shown. Magnification, ?63. Bars, 10 ?m.
VOL. 85, 2011 LCMV NP DOMAIN INVOLVED IN NP-Z INTERACTION13041
firm the reconstitution of YFP, we used a monoclonal antibody
that recognizes only the reconstituted ternary fluorescent
structure of YFP (Fig. 2C, ?-GFP). Consistent with our pre-
vious data using the M2H assay (Fig. 1), LCMV NP-Z inter-
action was detected when LCMV NP was fused to the N
terminus of EYN or EYC and LCMV Z was fused to the C
terminus of EYN or EYC. In contrast, we did not detect
LCMV NP-Z interaction in the other combinations, which
further supported the specificity of this NP-Z interaction.
Subcellular colocalization of LCMV NP and LCMV Z. Re-
sults from BiFC showed that reconstituted YFP fluorescence
mediated by NP-Z interactions was restricted to the cell cyto-
plasm (Fig. 2C). To confirm that NP-Z interaction occurred, as
predicted, in the cell cytoplasm, we cotransfected Vero cells
with LCMV NP-HA and LCMV Z-FLAG pCAGGs-based
expression plasmids alone or in combination (Fig. 3). Empty
pCAGGs plasmid was added in the single transfections to
normalize the amount of total transfected DNA. C-terminal
epitope tagging of both LCMV NP and Z proteins has previ-
ously been shown to not affect the function of these proteins
(40, 50). When expressed individually, both LCMV NP and Z
were distributed diffusely throughout the cytoplasm, but Z also
exhibited clear plasma membrane localization (Fig. 3A). Inter-
estingly, in cells expressing both NP and Z, we observed the
formation of cytoplasm aggregates where both NP and Z co-
localized. We next examined whether the formation of these
NP- and Z-containing inclusion bodies also occurred in
LCMV-infected cells. To that end, we infected Vero cells with
LCMV (MOI ? 0.1 PFU/cell), and at 48 hpi we examined the
cells by double immunofluorescence staining using an anti-NP
mouse monoclonal antibody and an anti-Z rabbit polyclonal
serum (Fig. 3B). We observed colocalization of NP and Z
within cytoplasmic inclusion bodies.
Mapping regions within LCMV NP required for its interac-
tion with LCMV Z. To identify the LCMV NP domains in-
volved in NP-Z interaction, we used a series of previously
described N-terminal (Fig. 4) and C-terminal (Fig. 5) LCMV
NP deletion mutants (38). We fused these mutants to VP16
FIG. 3. Subcellular colocation of LCMV NP and Z proteins. (A) NP-Z colocalization in transfected cells. Vero cells were cotransfected with
1 ?g of the indicated plasmids. Empty pCAGGs plasmid was used to normalize single plasmid transfections. At 48 h posttransfection, cells were
examined by immunofluorescence staining with the anti-HA (?-HA) monoclonal (NP staining) and anti-FLAG (?-FLAG) polyclonal (Z staining)
antibodies. Cellular nuclei were stained with DAPI. Merged and magnified squares are illustrated. (B) NP-Z colocalization in LCMV-infected cells.
Vero cells were mock or LCMV infected (MOI ? 0.1). At 48 hpi, subcellular localization of NP and Z was assessed using an anti-NP (?-NP)
monoclonal antibody (1.1.3) and an anti-Z (?-Z) polyclonal antibody. Merged images from NP (green), Z (red), and DAPI (blue) staining and
magnified images of the indicated squares are also illustrated. Representative images are shown. Magnification, ?63. Bars, 5 ?m.
13042 ORTIZ-RIAN ˜O ET AL. J. VIROL.
and used them in the M2H assay to assess their ability to
interact with Z.
Deletion of the N-terminal first 300 amino acids of LCMV
NP (Fig. 4A) did not significantly affect the ability of LCMV
NP to interact with LCMV Z (Fig. 4B). However, further
N-terminal deletions (e.g., ?N350) disrupted the ability of
LCMV NP to interact with LCMV Z (Fig. 4B). We observed
differences in expression levels among the NP N-terminal de-
letion mutants, but all were detected by Western blotting using
an anti-VP16 polyclonal antibody (Fig. 4C). It should be noted
that mutant ?N350 was impaired in its ability to interact with
Z but ?N350 was expressed at levels similar to NP wild type.
Likewise, we also examined the ability of LCMV NP C-termi-
nal deletion mutants to interact with LCMV Z (Fig. 5A).
Deletion of the last 5 amino acids did not affect the ability of
LCMV NP to interact with LCMV Z (Fig. 5B). However,
deletion of more than 5 amino acids in the C terminus of
LCMV NP (e.g., ?C10) significantly affected its interaction
with LCMV Z (Fig. 5B). This lack of interaction was not due
to significant differences in protein expression since all LCMV
NP C-terminal deletion mutants were expressed similarly to
wild-type LCMV NP (Fig. 5C), a finding consistent with pre-
viously published data (47). These results indicated that the
C-terminal region (amino acids 300 to 553) of LCMV NP is
required and sufficient for its interaction with LCMV Z.
Examining the relationship between anti-IFN activity of
LCMV NP and its ability to interact with LCMV Z. We have
previously shown that the C-terminal region of LCMV NP
(amino acids 370 to 553) is required for its anti-IFN activity
(38). Since the C-terminal domain of NP is also involved in its
interaction with LMCV Z, we examined whether both NP
activities could be separated in the primary structure of LCMV
NP. Support for the feasibility of segregating these two NP
activities was provided by the observation that TCRV NP lacks
the ability, compared to other arenavirus NPs, to counteract
the IFN response (39) and the rescue of a viable recombinant
LCMV carrying a D382A mutation in NP (rLCMVD382A)
that lacks the ability to counteract the IFN response (38), but
both TCRV NP-Z and LCMV NP-Z interactions are required
for production of infectious TCRV and LCMV, respectively.
FIG. 4. The N-terminal 300 amino acids of LCMV NP are not
required for NP-Z interaction. (A) Schematic representation of
LCMV NP wild type and N-terminal deletion mutants used in the
M2H assay system. Total amino acid lengths of wild type and NP
deletion mutants are indicated on the right. (B) LCMV NP-Z inter-
action with N-terminal deletion mutants. Cells (293T) were cotrans-
fected, and the presence of NP-Z interaction was quantified in cell
lysates as described in the legend to Fig. 1. Percentage of interaction of
N-terminal deletion mutants was calculated on the basis of wild-type
NP-Z interaction. (C) Protein expression levels of LCMV NP N-ter-
minal deletion mutants. Cell lysates from transfected 293T cells were
analyzed for protein expression levels by Western blotting using an
anti-VP16 (?-VP16) polyclonal antibody. GAPDH was used as a load-
ing control. Protein molecular mass markers (kDa) are indicated on
FIG. 5. The C-terminal domain of LCMV NP is involved in NP-Z
interaction. (A) Schematic representation of the LCMV NP C-termi-
nal deletion mutants used in the M2H assay system. Total amino acid
lengths of wild-type and NP C-terminal deletion mutants are indicated
on the right. (B) Role of NP C terminus on LCMV NP-Z interaction.
293T cells were cotransfected with the indicated plasmids, and NP-Z
interaction was quantified in cell lysates as described in the legend to
Fig. 1. Percentage of interaction of C-terminal deletion mutants was
calculated on the basis of wild-type NP-Z interaction. (C) Protein
expression levels of LCMV NP C-terminal deletion mutants. The same
cell extracts used for the experiment whose results are shown in panel
B were used to detect expression of LCMV NP wild-type and C-ter-
minal deletion mutants by Western blotting using a polyclonal anti-
VP16 (?-VP16) antibody. GAPDH expression levels were used as a
loading control. Protein molecular mass markers (kDa) are indicated
on the left.
VOL. 85, 2011 LCMV NP DOMAIN INVOLVED IN NP-Z INTERACTION13043
To experimentally test this hypothesis, we assessed in the M2H
assay the ability of LCMV NPs with mutations in the DIEGR
motif (D382A, G385A, and R386A) (38) and the recently
described 3?–5? exonuclease motif (D382A, E384A, D459A,
H517A, and D552A) (25, 54), all of which were previously
shown to play a critical role in the IFN-counteracting activity of
NP, to interact with Z (Fig. 6). As a control, we used the
LCMV NP I383A mutant that we previously showed retains its
anti-IFN function (38). All of the NP mutants interacted with
LCMV Z to levels comparable to those for the LCMV NP wild
type (Fig. 6A) and were expressed at levels comparable to
those for the LCMV NP wild type (Fig. 6B). These results
revealed that residues playing key roles in the anti-IFN activity
of NP are not critical for NP-Z interaction.
Assessing homotypic and heterotypic NP-Z interactions be-
tween LCMV and LASV. Coinfection of the same cells with two
phenotypically different arenavirus strains (or variants) results
in the generation of virus progeny containing reassortant vi-
ruses where S and L genome segments are exchanged. This
genetic approach has been widely used to define the function
of viral gene products in arenavirus pathogenesis. However, to
produce a viable reassortant arenavirus, proteins encoded by
the L and S genome segments of the two viral strains coinfect-
ing the same cell should permit the virus protein-protein in-
teractions required for production and multiplication of infec-
tious viral progeny. Arenavirus NP and Z proteins are encoded
by the S and L genome segments, respectively, and need to
interact to allow incorporation of vRNPs into Z-mediated bud-
ding viruses. Generation of reassortant viruses between OWAs
has been previously described (36). To assess heterotypic NP-Z
interactions between LASV and LCMV, we first confirmed the
NP-Z interaction in LASV using the M2H assay system (Fig.
7). LCMV NP interacted with LASV Z, but LASV NP did not
interact with LCMV Z, suggesting a unidirectional interaction
between NP and Z proteins from LASV and LCMV.
Z has been documented to be the driving force of arenavirus
budding (50, 51, 61, 62). Accordingly, NP-Z interaction is pre-
dicted to play a critical role in packaging vRNPs into Z-medi-
ated budding viral particles. Therefore, we predicted that
LASV NP would not be incorporated into LCMV Z-induced
VLPs. To examine NP incorporation into Z-mediated produc-
tion of VLPs, we used a previously described VLP assay (33)
(Fig. 8). For this, we generated HA-tagged versions of LCMV
and LASV NP and FLAG-tagged versions of LCMV and
LASV Z that were cotransfected alone or in combination into
293T cells. Seventy-two hours after transfection, tissue culture
supernatants and cell lysates were collected. Supernatants were
pelleted through a 20% sucrose cushion, and VLP-containing
FIG. 6. Critical amino acid residues required for the anti-IFN func-
tion of LCMV NP are not required for NP-Z interaction in the M2H
assay. 293T cells were cotransfected as described in the legend to Fig.
1 but using the indicated LCMV NP IFN mutants. At 48 h posttrans-
fection, NP-Z interaction was quantified by dual-luciferase reporter
assay. (A) Percentage of interaction of single amino acid mutants was
calculated on the basis of wild-type NP-Z interaction. (B) Protein
expression levels of LCMV NP mutants. The same cell lysates from the
experiment whose results are shown in panel A were used to detect
expression of LCMV NP wild type and single amino acid mutants by
Western blotting using an anti-VP16 (?-VP16) polyclonal antibody.
GAPDH expression levels were used as loading controls. Protein mo-
lecular mass markers (kDa) are indicated on the left.
FIG. 7. Assessing homotypic and heterotypic NP-Z interactions be-
tween LCMV and LASV by the M2H assay approach. 293T cells were
cotransfected, as described in the legend to Fig. 1, with the indicated
LCMV and LASV NP and Z mammalian two-hybrid expression plas-
mids. At 48 h posttransfection, homologous and heterologous NP-Z
protein interactions were detected by GFP expression using fluores-
cence microscopy (A). GAL4 and VP16 expression plasmids were
included as negative controls. Renilla luciferase values (means ? stan-
dard deviations) are indicated in each image. To quantify interactions,
cell lysates were prepared to detect luciferase expression, as described
in the legend to Fig. 1. (B) Percentage of interaction for heterologous
NP-Z interactions were normalized on the basis of homologous inter-
13044ORTIZ-RIAN ˜O ET AL.J. VIROL.
pellets were analyzed by Western blotting. We observed incor-
poration of both LCMV and LASV NPs into LASV Z-induced
VLPs. In contrast, LCMV NP but not LASV NP was incorpo-
rated into LCMV Z-mediated VLPs. As expected, expression
of LCMV or LASV NPs alone was not detected in the tissue
culture supernatants, demonstrating specific NP incorporation
in Z-induced VLPs. All tagged proteins were expressed to
similar levels, as determined by Western blotting of cell lysates
(Fig. 8, Input, ?-HA and ?-FLAG).
Colocalization of LCMV and LASV NP-Z. Since the ob-
served unidirectional NP-Z heterotypic interaction was unex-
pected, we attempted to confirm these results by assessing
colocalization of LCMV and LASV NP and Z proteins in
plasmid-transfected cells (Fig. 9). We cotransfected Vero cells
with HA-tagged LCMV and LASV NP with FLAG-tagged
LCMV and LASV Z alone or in different combinations.
LCMV NP (Fig. 9A) and LASV NP (Fig. 9B) showed a diffuse
distribution in the cytoplasm of transfected cells, whereas
LCMV Z (Fig. 9C) and LASV Z (Fig. 9D) were distributed
throughout the cytoplasm but were also present in the cell
membrane. Cotransfection of NP and Z from LCMV (Fig. 9E)
or from LASV (Fig. 9H) resulted in the formation in the cell
cytoplasm of inclusion bodies that contained both NP and Z
proteins. We observed similar inclusion bodies when we re-
placed LCMV Z by LASV Z in the presence of LCMV NP
(Fig. 9F). On the other hand, coexpression of LASV NP and
LCMV Z resulted in minimal colocalization of both viral pro-
teins and the absence of the large inclusion bodies (Fig. 9G),
confirming our results with the M2H and VLP assays.
In this work we have documented and initially characterized
the interaction between NP and Z proteins of the prototypic
arenavirus LCMV and the HF arenavirus LASV. The robust-
ness of LCMV NP-Z interaction was reflected by its detection
using three different approaches: M2H assay (Fig. 1), BiFC
(Fig. 2), and colocalization studies using double immunofluo-
rescence (Fig. 3). In addition, we have presented evidence that
LCMV NP and LASV Z, but not LASV NP and LCMV Z,
interact. This finding would suggest that heterotypic interac-
tions between NP and Z have a unidirectional component,
which could influence the generation of viable reassortant are-
naviruses. Using a collection of N-terminal (Fig. 4) and C-ter-
minal (Fig. 5) deletion mutants of LCMV NP, we identified the
C-terminal region (amino acids 300 to 553) of LCMV NP to be
responsible for interaction with LCMV Z. As the same C-ter-
minal region of LCMV NP is also involved in the anti-IFN
activity of NP (38), we examined the possibility that the anti-
IFN- and Z-binding activities of NP were linked and could not
be segregated. For this we assessed the Z-binding activity of a
collection of NP mutants with mutations within the conserved
DIEGR motif (D382A, G385A, R386) (38), as well as within
the active site (D382A, E384A, D459A, H517A, and D522A)
of the predicted 3?–5? exonuclease domain of LCMV NP on
the basis of findings reported for LASV NP (25, 54). We
selected these mutants because viruses with mutations within
the DIEGR motif, as well as those affecting the exonuclease
activity of NP, were found to be impaired in their anti-IFN
activity. Our results showed that NP mutants affected in their
anti-IFN function were still able to interact with LCMV Z to
levels comparable to those for the LCMV NP wild type (Fig.
6). These results indicated that different amino acid residues
govern these two NP activities. Additional support for this
conclusion stems from the observation that TCRV NP lacks
the ability to counteract the IFN response (39) and the rescue
of a recombinant LCMV carrying the D382A mutation in NP
(rLCMVD382A) that resulted in the loss of the virus’s ability
to counteract the IFN response (38), whereas both TCRV
NP-Z and LCMV NP-Z interactions were required for pro-
duction of TCRV and LCMV infections, respectively. NP-Z
interaction has also been described for TCRV (34), but TCRV
NP does not counteract the IFN response (38), and thereby,
these previous studies did not assess a possible overlap be-
tween the anti-IFN activity of NP and its ability to interact with
Z. The identification of NP mutants affected in their ability to
interact with Z but not in their anti-IFN function would help to
validate this conclusion.
We observed that NP and Z colocalized within inclusion
bodies in the cell cytoplasm, resembling typical factories of
virus replication. Results from minigenome (MG) reporter
assays have shown that Z is not strictly required for arenavirus
RNA replication and gene transcription, but it is plausible that
MG-based assays may not entirely re-create all the regulatory
aspects of viral RNA synthesis taking place during the course
FIG. 8. Homologous and heterologous NP incorporation into
LCMV and LASV Z-induced VLPs. 293T cells (6-well plate format)
were cotransfected with 2 ?g of the indicated LCMV or LASV NP
(HA tagged) and Z (FLAG tagged) pCAGGs expression plasmids.
Empty pCAGGs MCS plasmid was included, in all cases, to normalize
the amount of transfected DNA. At 72 h posttransfection, cell extracts
were prepared and analyzed for protein expression levels (Input).
Supernatants from the same transfections were used for isolation of
VLPs. Expression levels of the different arenavirus NPs and Zs were
detected by Western blotting using an anti-HA (?-HA) monoclonal
antibody (NPs) or an anti-FLAG (?-FLAG) polyclonal antibody (Zs).
GAPDH was used as a loading control. Numbers at the bottom of each
Western blot lane represent the band intensities as a percentage of
homologous NP or Z expression levels.
VOL. 85, 2011 LCMV NP DOMAIN INVOLVED IN NP-Z INTERACTION 13045
of arenavirus natural infection. On the other hand, Z has been
shown to inhibit viral transcription and replication (12–14, 35),
and our findings would suggest the possibility that NP-Z inter-
action could sequester vRNP from sites of viral RNA synthesis
into budding particles, although an interaction of Z with L may
similarly be responsible for this Z-mediated inhibitory effect on
viral RNA synthesis (29).
As predicted, we also observed an interaction between
LASV NP and Z proteins. Notably, results from the M2H assay
(Fig. 7) as well as VLP assays (Fig. 8) and colocalization stud-
ies (Fig. 9) indicated that LCMV NP interacted with LCMV
and LASV Z protein, whereas LASV NP interacted with
LASV but not LCMV Z protein. This unidirectional hetero-
typic interaction between NP and Z of LCMV and LASV
suggests the presence of a conserved protein domain within
NPs of both LCMV and LASV but with specific amino acid
residues playing key roles in determining the specificity of this
protein-protein interaction. It is worth noting that character-
ization of reassortant viruses between OWAs MOPV and
LASV readily identified viruses containing the L segment of
FIG. 9. Assessing homotypic and heterotypic NP-Z colocalizations. Vero cells were cotransfected, as described in the legend to Fig. 3, with the
indicated protein expression plasmids (left). At 48 h posttransfection, cells were fixed and permeabilized before immunofluorescence with an
anti-HA (?-HA) monoclonal antibody (NPs) and with an anti-FLAG (?-FLAG) polyclonal antibody (Zs). Merged images from NP (green), Z
(red), and DAPI (blue) staining and magnified images of the merger are illustrated. Representative images are shown. Magnification, ?63. Bars,
13046ORTIZ-RIAN ˜O ET AL.J. VIROL.
MOPV and S segment of LASV, but reassortants containing
LASV L segment and MOPV S segment were not obtained
(36). Based on our results with LCMV and LASV NP and Z
proteins, we would suggest that MOPV Z protein can interact
efficiently with LASV NP but LASV Z is not able to interact
efficiently with MOPV NP. To our knowledge, no reassortants
between LCMV and LASV have yet been described. Our re-
sults would predict that only reassortants containing the S
segment from LCMV and the L segment from LASV, but not
the opposite combination, would be viable due to the inability
of LASV NP to interact with LCMV Z.
The recently described crystal structure of LASV NP sug-
gested the possible NP self-association through the N- to C-
terminal interactions (54). However, these structural predic-
tions do not appear to be compatible with the biochemical and
functional results that we and others have obtained, indicating
that NP-NP interaction occurs through the N-terminal domain
of NP (34, 47). Deletions of as few as 50 amino acids in the
N-terminal end of LCMV NP affected the NP-NP interaction
without affecting its anti-IFN activity, suggesting that mono-
mers of NP might counteract the IFN response and interact
with Z. This, in turn, raises the intriguing possibility that NP-
related smaller polypeptides reported in cells infected with
some arenaviruses, including LASV (11) and the NWA
Pichinde virus (22), could represent C-terminal fragments able
to interact with Z and also contribute to the virus anti-IFN
Based on our findings, we propose NP to be constituted by
two distinct domains (Fig. 10). The C-terminal domain (amino
acids 370 to 553) is responsible for counteracting the host type
I IFN response (38). Within this domain, the DIEGR (38) and
the 3?–5? exonuclease (25, 54) motifs would play critical roles
in the anti-IFN activity. However, arenaviruses with limited
anti-IFN activity, like TCRV NP (19, 39) and, possibly, MOPV
NP (37, 49), contained all the conserved residues of the pre-
dictive active site of the 3?-5? exonuclease motif (25, 38, 39, 54).
This could suggest that although the 3?–5? exonuclease activity
of NP may play an important role in counteracting the IFN
response, additional NP components are required for NP to
display fully its anti-IFN activity. In addition to its anti-IFN
activity, the C-terminal domain also directs the interaction of
NP with Z, but the key residues involved in this activity are
distinct from those contributing to the anti-IFN activity of NP.
The N-terminal domain (amino acids 1 to 358) is involved in
NP self-association (34, 47). None of these two domains can,
however, by itself, mediate either RNA replication or tran-
scription by the virus L polymerase, which requires the entire
integrity of NP, with the exception of its last five C-terminal
The significance of arenaviruses in human health and bio-
defense readiness, together with the limited existing armamen-
tarium to combat these infections, underscores the importance
of developing novel effective antiarenaviral drugs. To this end,
targeting NP-NP and NP-Z interactions represents a novel
antiarenaviral strategy. The development of assays and screen-
ing procedures to identify drugs to pursue this strategy could
be implemented without the need for the high biosafety con-
tainment required for the use of live virus in the case of highly
pathogenic arenaviruses, which would greatly facilitate the re-
search. Use of single-cycle infectious arenaviruses limited to
replication in GP-expressing cell lines may represent a safer
approach for the identification of antivirals targeting multiple
aspects of the replication cycle of arenaviruses (55). Our re-
sults also suggest that it might be possible to identify com-
pounds capable of disrupting NP-NP and NP-Z interactions
for a variety of arenaviruses, thus representing candidates for
the development of antiviral drugs with a activity against a
broad range of human-pathogenic arenaviruses.
We thank members of the L.M.-S. laboratory for their discussions,
especially to Shanaka Rodrigo and Snezhana Dimitrova for technical
advice and support. We also thank Juan Ayllon and Adolfo García-
Sastre (Mount Sinai School of Medicine) for providing EYN and EYC
expression plasmids and helpful advice with the BiFC assays. We also
show our appreciation to Alba Guarne (Health Sciences Center,
McMaster University) for her valuable comments and discussions.
E.O.-R. is a Fulbright-Conicyt fellowship recipient (BIO 2008). Re-
search in the L.M.-S. laboratory was partially funded by NIAID grant
RO1AI077719. Research by J.C.D.L.T. was supported by grants RO1
AI047140, RO1 AIO77719, and RO1 AI079665 from NIH/NIAID.
1. Barton, L. L. 1996. Lymphocytic choriomeningitis virus: a neglected central
nervous system pathogen. Clin. Infect. Dis. 22:197.
2. Battegay, M., et al. 1991. Quantification of lymphocytic choriomeningitis
virus with an immunological focus assay in 24- or 96-well plates. J. Virol.
3. Borden, K. L., E. J. Campbell Dwyer, and M. S. Salvato. 1998. An arenavirus
RING (zinc-binding) protein binds the oncoprotein promyelocyte leukemia
protein (PML) and relocates PML nuclear bodies to the cytoplasm. J. Virol.
4. Borio, L., et al. 2002. Hemorrhagic fever viruses as biological weapons:
medical and public health management. JAMA 287:2391–2405.
5. Borrow, P., L. Martinez-Sobrido, and J. C. de la Torre. 2010. Inhibition of
the type I interferon antiviral response during arenavirus infection. Viruses
6. Borrow, P., and M. B. Oldstone. 1994. Mechanism of lymphocytic chorio-
meningitis virus entry into cells. Virology 198:1–9.
FIG. 10. LCMV NP functional domains. Schematic representation
of LCMV NP primary structure indicating domains involved in protein
functions (top) and interactions (bottom). The entire primary se-
quence of LCMV NP is required for replication and transcription, with
the exception of the last 5 amino acids, on the basis of the results of a
minigenome reporter assay (38). The N-terminal domain (amino acids
1 to 358) contains the region involved in self-association (47). The
C-terminal domain contains the region responsible for counteracting
the IFN response (amino acids 370 to 553), including the DIEGR
motif (rectangle) (38) and the catalytic site of the 3?–5? exonuclease
motif (red) (25, 54), and the region involved in the interaction with
LCMV Z (amino acids 350 to 553).
VOL. 85, 2011LCMV NP DOMAIN INVOLVED IN NP-Z INTERACTION13047
7. Briese, T., et al. 2009. Genetic detection and characterization of Lujo virus,
a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS
8. Buchmeier, M. J., J. C. de la Torre, and C. J. Peters. 2007. Arenaviridae: the
viruses and their replication, p. 1791–1827. In P. D. Knipe et al. (ed.), Fields
virology, 5th ed., vol. II. Lippincott Williams & Wilkins, Philadelphia, PA.
9. Campbell Dwyer, E. J., H. Lai, R. C. MacDonald, M. S. Salvato, and K. L.
Borden. 2000. The lymphocytic choriomeningitis virus RING protein Z as-
sociates with eukaryotic initiation factor 4E and selectively represses trans-
lation in a RING-dependent manner. J. Virol. 74:3293–3300.
10. Charrel, R. N., and X. de Lamballerie. 2003. Arenaviruses other than Lassa
virus. Antiviral Res. 57:89–100.
11. Clegg, J. C., and G. Lloyd. 1983. Structural and cell-associated proteins of
Lassa virus. J. Gen. Virol. 64:1127–1136.
12. Cornu, T. I., and J. C. de la Torre. 2002. Characterization of the arenavirus
RING finger Z protein regions required for Z-mediated inhibition of viral
RNA synthesis. J. Virol. 76:6678–6688.
13. Cornu, T. I., and J. C. de la Torre. 2001. RING finger Z protein of lympho-
cytic choriomeningitis virus (LCMV) inhibits transcription and RNA repli-
cation of an LCMV S-segment minigenome. J. Virol. 75:9415–9426.
14. Cornu, T. I., H. Feldmann, and J. C. de la Torre. 2004. Cells expressing the
RING finger Z protein are resistant to arenavirus infection. J. Virol. 78:
15. Eichler, R., et al. 2004. Characterization of the Lassa virus matrix protein Z:
electron microscopic study of virus-like particles and interaction with the
nucleoprotein (NP). Virus Res. 100:249–255.
16. Fan, L., T. Briese, and W. I. Lipkin. 2010. Z proteins of New World arena-
viruses bind RIG-I and interfere with type I interferon induction. J. Virol.
17. Fischer, S. A., et al. 2006. Transmission of lymphocytic choriomeningitis
virus by organ transplantation. N. Engl. J. Med. 354:2235–2249.
18. Garcin, D., S. Rochat, and D. Kolakofsky. 1993. The Tacaribe arenavirus
small zinc finger protein is required for both mRNA synthesis and genome
replication. J. Virol. 67:807–812.
19. Groseth, A., et al. 2011. Tacaribe virus but not Junin virus infection induces
cytokine release from primary human monocytes and macrophages. PLoS
Negl. Trop. Dis. 5:e1137.
20. Groseth, A., S. Wolff, T. Strecker, T. Hoenen, and S. Becker. 2010. Efficient
budding of the Tacaribe virus matrix protein Z requires the nucleoprotein.
J. Virol. 84:3603–3611.
21. Gunther, S., and O. Lenz. 2004. Lassa virus. Crit. Rev. Clin. Lab. Sci.
22. Harnish, D. G., W. C. Leung, and W. E. Rawls. 1981. Characterization of
polypeptides immunoprecipitable from Pichinde virus-infected BHK-21
cells. J. Virol. 38:840–848.
23. Harrison, L. H., et al. 1999. Clinical case definitions for Argentine hemor-
rhagic fever. Clin. Infect. Dis. 28:1091–1094.
24. Harrison, M. S., T. Sakaguchi, and A. P. Schmitt. 2010. Paramyxovirus
assembly and budding: building particles that transmit infections. Int.
J. Biochem. Cell Biol. 42:1416–1429.
25. Hastie, K. M., C. R. Kimberlin, M. A. Zandonatti, I. J. MacRae, and E. O.
Saphire. 2011. Structure of the Lassa virus nucleoprotein reveals a dsRNA-
specific 3? to 5? exonuclease activity essential for immune suppression. Proc.
Natl. Acad. Sci. U. S. A. 108:2396–2401.
26. Holmes, G. P., et al. 1990. Lassa fever in the United States. Investigation of
a case and new guidelines for management. N. Engl. J. Med. 323:1120–1123.
27. Hu, C. D., and T. K. Kerppola. 2003. Simultaneous visualization of multiple
protein interactions in living cells using multicolor fluorescence complemen-
tation analysis. Nat. Biotechnol. 21:539–545.
28. Isaacson, M. 2001. Viral hemorrhagic fever hazards for travelers in Africa.
Clin. Infect. Dis. 33:1707–1712.
29. Jacamo, R., N. Lopez, M. Wilda, and M. T. Franze-Fernandez. 2003. Tac-
aribe virus Z protein interacts with the L polymerase protein to inhibit viral
RNA synthesis. J. Virol. 77:10383–10393.
30. Jahrling, P. B., and C. J. Peters. 1992. Lymphocytic choriomeningitis virus.
A neglected pathogen of man. Arch. Pathol. Lab. Med. 116:486–488.
31. Kilgore, P. E., et al. 1997. Treatment of Bolivian hemorrhagic fever with
intravenous ribavirin. Clin. Infect. Dis. 24:718–722.
32. Lee, K. J., I. S. Novella, M. N. Teng, M. B. Oldstone, and J. C. de La Torre.
2000. NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are
sufficient for efficient transcription and replication of LCMV genomic RNA
analogs. J. Virol. 74:3470–3477.
33. Lee,K.J.,M.Perez,D.D.Pinschewer,andJ.C.delaTorre. 2002.Identification
of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue
LCMV RNA analogs into LCMV like particles. J. Virol. 76:6393–6397.
34. Levingston Macleod, J. M., et al. 2011. Identification of two functional
domains within the arenavirus nucleoprotein. J. Virol. 85:2012–2023.
35. Lopez, N., R. Jacamo, and M. T. Franze-Fernandez. 2001. Transcription and
RNA replication of Tacaribe virus genome and antigenome analogs require
N and L proteins: Z protein is an inhibitor of these processes. J. Virol.
36. Lukashevich, I. S. 1992. Generation of reassortants between African arena-
viruses. Virology 188:600–605.
37. Lukashevich, I. S., et al. 1999. Lassa and Mopeia virus replication in human
monocytes/macrophages and in endothelial cells: different effects on IL-8
and TNF-alpha gene expression. J. Med. Virol. 59:552–560.
38. Martinez-Sobrido, L., et al. 2009. Identification of amino acid residues crit-
ical for the anti-interferon activity of the nucleoprotein of the prototypic
arenavirus lymphocytic choriomeningitis virus. J. Virol. 83:11330–11340.
39. Martinez-Sobrido, L., P. Giannakas, B. Cubitt, A. Garcia-Sastre, and J. C.
de la Torre. 2007. Differential inhibition of type I interferon induction by
arenavirus nucleoproteins. J. Virol. 81:12696–12703.
40. Martínez-Sobrido, L., E. I. Zu ´n ˜iga, D. Rosario, A. García-Sastre, and J. C.
de la Torre. 2006. Inhibition of the type I interferon response by the nucleo-
protein of the prototypic arenavirus lymphocytic choriomeningitis virus.
J. Virol. 80:9192–9199.
41. McCormick, J. B., and S. P. Fisher-Hoch. 2002. Lassa fever. Curr. Top.
Microbiol. Immunol. 262:75–109.
42. McCormick, J. B., et al. 1986. Lassa fever. Effective therapy with ribavirin.
N. Engl. J. Med. 314:20–26.
43. McKee, K. T., Jr., J. W. Huggins, C. J. Trahan, and B. G. Mahlandt. 1988.
Ribavirin prophylaxis and therapy for experimental Argentine hemorrhagic
fever. Antimicrob. Agents Chemother. 32:1304–1309.
44. Mets, M. B., L. L. Barton, A. S. Khan, and T. G. Ksiazek. 2000. Lymphocytic
choriomeningitis virus: an underdiagnosed cause of congenital chorioretini-
tis. Am. J. Ophthalmol. 130:209–215.
45. Munoz-Jordan, J. L., et al. 2005. Inhibition of alpha/beta interferon signaling
by the NS4B protein of flaviviruses. J. Virol. 79:8004–8013.
46. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-
expression transfectants with a novel eukaryotic vector. Gene 108:193–199.
47. Ortiz-Rian ˜o, E., B. Cheng, J. C. de la Torre, and L. Martinez-Sobrido.
Self-association of lymphocytic choriomeningitis virus nucleoprotein is me-
diated by its N-terminal region and is not required for its anti-interferon
function. J. Virol., in press.
48. Palacios, G., et al. 2008. A new arenavirus in a cluster of fatal transplant-
associated diseases. N. Engl. J. Med. 358:991–998.
49. Pannetier, D., C. Faure, M. C. Georges-Courbot, V. Deubel, and S. Baize.
2004. Human macrophages, but not dendritic cells, are activated and pro-
duce alpha/beta interferons in response to Mopeia virus infection. J. Virol.
50. Perez, M., R. C. Craven, and J. C. de la Torre. 2003. The small RING finger
protein Z drives arenavirus budding: implications for antiviral strategies.
Proc. Natl. Acad. Sci. U. S. A. 100:12978–12983.
51. Perez, M., D. L. Greenwald, and J. C. de la Torre. 2004. Myristoylation of the
RING finger Z protein is essential for arenavirus budding. J. Virol. 78:
52. Peters, C. J. 2002. Human infection with arenaviruses in the Americas. Curr.
Top. Microbiol. Immunol. 262:65–74.
53. Pinschewer, D. D., M. Perez, and J. C. de la Torre. 2003. Role of the virus
nucleoprotein in the regulation of lymphocytic choriomeningitis virus tran-
scription and RNA replication. J. Virol. 77:3882–3887.
54. Qi, X., et al. 2010. Cap binding and immune evasion revealed by Lassa
nucleoprotein structure. Nature 468:779–783.
55. Rodrigo, W. W., J. C. de la Torre, and L. Martinez-Sobrido. 2011. Use of
single-cycle infectious lymphocytic choriomeningitis virus to study hemor-
rhagic fever arenaviruses. J. Virol. 85:1684–1695.
56. Rodriguez, M., J. B. McCormick, and M. C. Weissenbacher. 1986. Antiviral
effect of ribavirin on Junin virus replication in vitro. Rev. Argent. Microbiol.
57. Rossman, J. S., and R. A. Lamb. 2011. Influenza virus assembly and budding.
58. Schmitt, A. P., and R. A. Lamb. 2004. Escaping from the cell: assembly and
budding of negative-strand RNA viruses. Curr. Top. Microbiol. Immunol.
59. Shtanko, O., et al. 2010. A role for the C terminus of Mopeia virus nucleo-
protein in its incorporation into Z protein-induced virus-like particles. J. Vi-
60. Snell, N. 1988. Ribavirin therapy for Lassa fever. Practitioner 232:432.
61. Strecker, T., et al. 2003. Lassa virus Z protein is a matrix protein and
sufficient for the release of virus-like particles [corrected]. J. Virol. 77:10700–
62. Strecker, T., et al. 2006. The role of myristoylation in the membrane asso-
ciation of the Lassa virus matrix protein Z. Virol. J. 3:93.
63. Urata, S., J. Yasuda, and J. C. de la Torre. 2009. The Z protein of the New
World arenavirus Tacaribe virus has bona fide budding activity that does not
depend on known late domain motifs. J. Virol. 83:12651–12655.
64. Weissenbacher, M. C., R. P. Laguens, and C. E. Coto. 1987. Argentine
hemorrhagic fever. Curr. Top. Microbiol. Immunol. 134:79–116.
65. Wilda, M., N. Lopez, J. C. Casabona, and M. T. Franze-Fernandez. 2008.
Mapping of the Tacaribe arenavirus Z-protein binding sites on the L protein
identified both amino acids within the putative polymerase domain and a
region at the N terminus of L that are critically involved in binding. J. Virol.
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