By using a reverse genetics system that is based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we have identified the arenavirus small RING finger Z protein as the main driving force of virus budding. Both LCMV and Lassa fever virus (LFV) Z proteins exhibited self-budding activity, and both substituted efficiently for the late domain that is present in the Gag protein of Rous sarcoma virus. LCMV and LFV Z proteins contain proline-rich motifs that are characteristic of late domains. Mutations in the PPPY motif of LCMV Z severely impaired the formation of virus-like particles. LFV Z contains two different proline-rich motifs, PPPY and PTAP, which are separated by eight amino acids. Mutational analysis revealed that both motifs are required for efficient LFV Z-mediated budding. Both LCMV and LFV Z proteins recruited to the plasma membrane Tsg101, which is a component of the class E vacuolar protein sorting machinery that has been implicated in budding of HIV and Ebola virus. A renaviruses include Lassa fever virus (LFV) and the South American hemorrhagic fever (HF) viruses. These viruses cause severe human disease, and they pose a threat as agents of bioterrorism (1). The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is an important model with which to study both acute and persistent viral infection (2). In addition, LCMV provides an excellent system with which to study basic aspects of the molecular and cell biology of HF arenaviruses.LCMV is an enveloped virus, whose genome consists of two negative-sense, single-stranded RNA segments, called L (7.2 kb) and S (3.4 kb). Each segment uses an ambisense coding organization to direct the synthesis of two gene products in opposite orientation, and each is separated by an intergenic region (3) The S RNA encodes the nucleoprotein (NP), and the two surface virion glycoproteins (GPs), GP-1 and GP-2, which are derived by proteolytic cleavage of a precursor polypeptide, GP-C (4). GP-1 and GP-2 form the spikes on the virion envelope and mediate cell entry by interaction with the host cell surface receptor (5). The L RNA directs the synthesis of the virus RNA-dependent RNA polymerase (L protein), and a small RING finger protein called Z (11 kDa) (6). The NP associates with the viral genomic RNA species and L to form the viral ribonucleoprotein (RNP) core that is competent in transcription and RNA replication, and constitutes the minimal infectious unit (7). The role of Z in the virus life cycle is poorly understood, and homologues of Z are not found in other negativestrand (NS) RNA viruses. Z is a structural component of the virus (8), and in infected cells, Z has been reported to interact with several cellular factors, including promyelocytic leukemia protein (9), and the eukaryotic translation initiation factor 4E, the latter of which has been proposed to repress CAP-dependent translation (10, 11). In addition, early studies suggested a role of Z in viral transcriptional regulation (12).We have developed a reverse genetic system f...

The arenavirus small RING finger Z protein is the main driving force of arenavirus budding. The primary structure of Z is devoid of hydrophobic transmembrane domains, but both lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus Z proteins accumulate near the inner surface of the plasma membrane and are strongly membrane associated. All known arenavirus Z proteins contain a glycine (G) at position 2, which is a potential acceptor site for a myristoyl moiety. Metabolic labeling showed incorporation of [ 3 H]myristic acid by wild-type Z protein but not by the G2A mutant. The mutation G2A eliminated Z-mediated budding. Likewise, treatment with the myristoylation inhibitor 2-hydroxymyristic acid inhibited Z-mediated budding, eliminated formation of virus-like particles, and caused a dramatic reduction in virus production in LCMVinfected cells. Budding activity was restored in G2A mutant Z proteins by the addition of the myristoylation domain of the tyrosine protein kinase Src to their N termini. These findings indicate N-terminal myristoylation of Z plays a key role in arenavirus budding.

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as ␣-dystroglycan (␣-DG), a cell surface receptor that provides a molecular link between the extracellular matrix and the actin-based cytoskeleton. In the present study, we show that LFV binds to ␣-DG with high affinity in the low-nanomolar range. Recombinant vesicular stomatitis virus pseudotyped with LFV glycoprotein (GP) adopted the receptor binding characteristics of LFV and depended on ␣-DG for infection of cells. Mapping of the binding site of LFV on ␣-DG revealed that LFV binding required the same domains of ␣-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin ␣1 and ␣2 chains for ␣-DG binding. Together with our previous studies on receptor binding of the prototypic immunosuppressive LCMV isolate LCMV clone 13, these findings indicate a high degree of conservation in the receptor binding characteristics between the highly human-pathogenic LFV and murine-immunosuppressive LCMV isolates.

Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3 ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome. Each segment uses an ambisense coding strategy to direct the synthesis of two gene products. Besides lymphocytic choriomeningitis virus (LCMV), its prototype member, the arenavirus family comprises important human pathogens, such as Lassa fever virus (LFV) and the South American hemorrhagic fever viruses. The short (S; 3.4-kb) (31) segment of LCMV encodes the viral glycoprotein (GP) precursor (GP-C; 75 kDa) and nucleoprotein (NP; ca. 63 kDa). The large (L; 7.2-kb) (30, 32) segment carries the open reading frames (ORF) for the viral RNA-dependent RNA polymerase (RdRp) gene L (ca. 200 kDa) and for the small zinc finger protein Z (11 kDa). NP encapsidates the genome to generate the template recognized by the virus polymerase. NP and L represent the minimal viral transacting factors required for viral RNA transcription and replication (14), whereas Z is the main driving force of arenavirus budding (23). Studies using minireplicon systems for the Old World arenavirus LCMV (5, 6) as well as for the New World arenavirus Tacaribe virus (TV; 18) have revealed an inhibitory activity of Z on virus transcription and replication. Consistent with this finding, cells expressing LCMV or LFV Z proteins are refractory to infection with LCMV or LFV, respe...

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor ␣-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has a bisegmented negative-strand RNA genome. Each segment carries two viral genes in opposite orientation and separated by an intergenic region (IGR). The RNA-dependent RNA polymerase (RdRp) L of LCMV produces subgenomic mRNA and full-length genomic and antigenomic RNA species in two different processes termed transcription and replication, respectively. It is widely accepted that intracellular nucleoprotein (NP) levels regulate these two processes. Intracellular NP levels increase during the course of the infection, resulting in the unfolding of secondary RNA structures within the IGR. Structure-dependent transcription termination at the IGR is thereby attenuated, promoting replication of genome and antigenome RNA species. To test this hypothesis, we established a helper-virus-free minigenome (MG) system where intracellular synthesis of an S segment analogue from a plasmid is driven by RNA polymerase I. Cotransfection with two additional plasmids expressing the minimal viral trans-acting factors L and NP under control of RNA polymerase II allowed for RNA synthesis mediated by the intracellularly reconstituted LCMV polymerase. Both processes, transcription and replication, were strictly dependent on NP. However, both were equally enhanced by incrementally increasing amounts of NP up to levels in the range of those in LCMV-infected cells. Our data are consistent with a central role for NP in transcription and replication of the LCMV genome, but they do not support the participation of NP levels in balancing the two processes.The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an enveloped virus with a bisegmented, negative-strand RNA genome. The two genomic RNA segments are called large (L) and short (S) and have sizes of 7.2 and 3.4 kb, respectively (27-29). Each segment directs the synthesis of two gene products, encoded in ambisense orientation. The two open reading frames of each segment are separated by an intergenic region (IGR), with a sequence predicted to form a hairpin structure of high stability (29). The S segment encodes the virus nucleoprotein (NP; ca. 63 kDa) and glycoprotein (GP) precursor, whereas the L segment encodes a large polypeptide (L protein; ca. 200 kDa) and a small RING finger protein (Z; ca. 11 kDa), whose function in the virus life cycle is poorly understood (29). The NP and L genes are carried in antigenome polarity 3Ј of the IGR in the S and L segments, respectively. NP encapsidates the genome segments and represents the most abundant protein in virions and infected cells (2). The L protein (ca. 200 kDa) exhibits the characteristic hallmarks common to the RNA-dependent RNA polymerases (RdRp) of negative-strand RNA viruses (27). GP and Z are encoded in genome polarity 5Ј of the IGR in the S and L segments, respectively. GP is synthesized as precursor polypeptide GP-C (75 kDa), which is posttranslationally processed into GP-1 (40 to 46 kDa) and GP-2 (35 kDa) (3). These two proteins remain noncovale...

The genome of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense, single-strand RNA segments designated L and S. Arenavirus genomes exhibit high sequence conservation at their 3 ends. All arenavirus genomes examined to date have a conserved terminal sequence element (3-terminal 20 nucleotides [nt]) thought to be a highly conserved viral promoter. Terminal complementarity between the 5 and 3 ends of the L and S RNAs predicts the formation of a thermodynamically stable panhandle structure that could contribute to the control of RNA synthesis. We investigated these issues by using a transcription-and replication-competent minireplicon system. A series of overlapping deletions spanning the 3-terminal 20-nt region of an LCMV minigenome (MG) was generated, and the mutant MGs were analyzed for their activity as templates for RNA synthesis by the LCMV polymerase. The minimal LCMV genomic promoter was found to be contained within the 3-terminal 19 nt. Substitution of C for G at the last 3-end nucleotide position in the MG resulted in nondetection of RNA transcription or replication, whereas the addition of a C at the 3 end did not have any significant affect on RNA synthesis mediated by the LCMV polymerase. All other mutations introduced within the 3-terminal 19 nt of the MG resulted in undetectable levels of promoter activity. Deletions and nucleotide substitutions within the MG 5 end that disrupted terminal complementarity abolished chloramphenicol acetyltransferase expression and RNA synthesis mediated by the LCMV polymerase. These findings indicate that both sequence specificity within the 3-terminal 19 nt and the integrity of the predicted panhandle structure appear to be required for efficient RNA synthesis mediated by the LCMV polymerase.The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is one of the most widely used model systems to study virus-host interactions, such as viral persistence and associated disease (8, 39). The LCMV genome is composed of two negative-sense single-stranded RNA segments, called S (3.2 kb) and L (7.2 kb) (46, 49). Both segments use an ambisense coding strategy to direct synthesis of two proteins from two open reading frames with opposite orientation and separated by an intergenic region (IGR) (2, 3, 60). The S RNA encodes the nucleoprotein NP (ca. 63 kDa) and the glycoprotein precursor GP-C (75 kDa). GP-C is posttranslationally cleaved to yield the mature glycoproteins GP-1 (40 to 46 kDa) and GP-2 (35 kDa) (45,54,62). Tetramers of GP-1 and GP-2 form the spikes on the virion envelope and mediate virus interaction with the cellular receptor (9, 11). The L RNA encodes the virus RNA-dependent RNA polymerase (RdRp) (L, ca. 200 kDa) (21, 30, 51) and a small (11-kDa) RING finger protein (Z) (49). NP and L are associated with the viral RNA to form ribonucleoprotein (RNP) complexes, which are active in transcription and replication (14,22). As with other negative-strand RNA viruses, this RNP is the minimal infectious unit.All cis-...

T he prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model to study both acute and persistent viral infection, as well as virus-host balance (1) and associated disease (2). Important concepts of immunology (3) and viral pathogenesis (4) have been developed by using the LCMV model. In addition, LCMV provides an excellent system to study basic aspects of the molecular and cell biology of clinically important human pathogens, including Lassa fever virus (LFV) and other arenaviruses causing severe hemorrhagic fever.LCMV is an enveloped bisegmented negative-strand RNA virus. The two genome segments L and S have approximate sizes of 7.2 and 3.4 kb, respectively (5, 6). Each segment uses an ambisense strategy to direct the synthesis of two proteins in opposite orientations, separated by an intergenic region. The S RNA contains the nucleoprotein (NP) and the glycoprotein (GP) precursor (GPC) genes, which are encoded in antigenome and genome polarity, respectively. Posttranslational processing of GPC produces GP-1 and -2 (7) and was recently shown to be mediated by the cellular protease S1P (8). GP-1 and -2 make up the spikes on the virion envelope and mediate cell entry by interaction with the host cell surface receptor. The L RNA segment codes for the virus RNA-dependent RNA polymerase (L) and a small (11-kDa) RING finger protein (Z), whose role in the virus life cycle is poorly understood.The inability to genetically manipulate the virus genome has hampered studies aimed at understanding the molecular and cell biology of LCMV. We have described a LCMV minigenome (MG) rescue system based on the use of reverse genetic approaches (9-11). We have identified NP and L as the minimal transacting viral factors required for virus replication and transcription. Moreover, we found that the viral 5Ј and 3Ј UTR, together with the intergenic region, are sufficient cis-acting signals for RNA synthesis by the LCMV RNA-dependent RNA polymerase. Z was not required for MG transcription and replication and exhibited a dose-dependent inhibitory effect on both processes (12). We have also shown that assembly and budding of LCMV infectious virus-like particles (VLPs) require both GP and Z (10).