Arenavirus Lymphocytic Choriomeningitis Virus Research Articles

Inhibition of the Type I Interferon Response by the Nucleoprotein of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-beta) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.

Rescue of the prototypic Arenavirus LCMV entirely from plasmid

We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5′- and 3′-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7RP. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome.

RNA Interference-Mediated Virus Clearance from Cells both Acutely and Chronically Infected with the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.

Mutagenesis-induced, large fitness variations with an invariant arenavirus consensus genomic nucleotide sequence.

Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.

Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5' untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.

Genetic and Biochemical Evidence for an Oligomeric Structure of the Functional L Polymerase of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

The arenavirus L protein has the characteristic sequence motifs conserved among the RNA-dependent RNA polymerase L proteins of negative-strand (NS) RNA viruses. Studies based on the use of reverse-genetics approaches have provided direct experimental evidence of the key role played by the arenavirus L protein in viral-RNA synthesis. Sequence alignment shows six conserved domains among L proteins of NS RNA viruses. The proposed polymerase module of L is located within its domain III, which contains highly conserved amino acids within motifs designated A and C. We have examined the role of these conserved residues in the polymerase activity of the L protein of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), in vivo using a minigenome rescue assay. We show here that the presence of sequence SDD, a characteristic of motif C of segmented NS RNA viruses, as well as the presence of the highly conserved D residue within motif A of L proteins, is strictly required for the polymerase activity of the LCMV L protein. The strong dominant negative phenotype associated with many of the mutants examined and results from coimmunoprecipitation studies provided genetic and biochemical evidence, respectively, for the requirement of the L-L interaction for the polymerase activity of the LCMV L protein.

Characterization of the Interaction of Lassa Fever Virus with Its Cellular Receptor α-Dystroglycan

The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as alpha-dystroglycan (alpha-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 alpha-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 alpha-DG for infection of cells. Mapping of the binding site of LFV on alpha-DG revealed that LFV binding required the same domains of alpha-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin alpha1 and alpha2 chains for alpha-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.

Dual Role of the Lymphocytic Choriomeningitis Virus Intergenic Region in Transcription Termination and Virus Propagation

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.

Arenavirus extinction through lethal mutagenesis

Viral hemorrhagic fevers represent serious human public health problems causing devastating and often lethal disease. Several hemorrhagic fevers are caused by arenaviruses including Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In recent years, increased air travel between Africa and other areas has led to the importation of LFV into the US, Europe, Japan, and Canada. This has raised awareness about arenaviruses as potential emerging viruses. Moreover, because of its severe morbidity and high mortality, and transmissibility from human to human, weaponized forms of LFV poses a real threat as agent of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these infections. Therefore, the importance of developing novel effective antiviral drugs to combat HF arenaviruses, for which the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) provides us with an excellent model system. Recent findings have shown that LCMV multiplication both in cultured cells and in vivo is highly susceptible to the mutagenic agent 5-fluorouracil (FU). FU-mediated extinction of LCMV was associated with only modest increases in virus mutation frequencies, but did not significantly affect virus replication and transcription, or virus particle formation. These findings indicate that, as with other riboviruses, lethal mutagenesis is effective also against LCMV raising the possibility of using this novel antiviral strategy to combat pathogenic arenaviruses.

491. Improved Apical Airway Epithelia Targeting Properties of FIV Vector Pseudotyped with Envelopes from LCMV

Gene transfer to the airway epithelia can correct the ion transport and host defense defects associated with cystic fibrosis (CF). However, the clinical application of gene therapy for CF is limited by several problems including adverse immune responses, inefficient transduction, and the failure of gene expression to persist. We hypothesized that the transduction efficiency of a (feline immunodeficiency virus) FIV-based lentivirus for the apical surface of polarized human airway epithelia (HAE) could be improved by pseudotyping the vector with glycoproteins from non-retroviral enveloped viruses. We found that glycoproteins from the arenavirus lymphocytic choriomeningitis virus (LCMV, WE54 strain) produced FIV titers similar to VSV-G FIV (108-109 TU/ml). Application of FIV pseudotyped with LCMV to well differentiated HAE yielded significantly more efficient gene transfer from the apical than the basolateral surface. Transduction by LCMV FIV from the apical surface was 3–4 fold more efficient than VSV-G FIV from the basolateral surface. Additionally, transduction was decreased by inhibitors of endosomal acidification, consistent with entry via a pH-sensitive endosomal pathway. Alpha-dystroglycan has been identified as a high affinity receptor for some arenavirus family members, including the WE54 strain of LCMV. By immunohistochemistry, alpha-dystroglycan was abundantly expressed on both the apical and basolateral surfaces of airway epithelia. Pretreatment of epithelia with laminin or antibodies to alpha-dystroglycan partially inhibited gene transfer with LCMV-FIV, suggesting that alpha-dystroglycan serves as a receptor in HAE. Vectors with a single point mutation to the LCMV WE54 envelope, designed to generate LCMV FIV virions with low affinity to alpha-dystroglycan, transduced airway epithelia 5–12 fold more efficiently from the apical surface than the high affinity WE54 glycoprotein pseudotyped virions. Transduction by the modified pseudotype was not inhibited by laminin or alpha-dystroglycan blocking antibody. Transduction levels with the low affinity alpha-dystroglycan binding envelope were also increased following basolateral application. These results with an envelope with low affinity for alpha-dystroglycan suggest that there is at least one additional unidentified viral receptor or cofactor for LCMV binding in airway epithelia. LCMV pseudotyped FIV provides a new tool to deliver transgenes to polarized airway epithelia via the apical surface.

Transduction Patterns of Pseudotyped Lentiviral Vectors in the Nervous System

We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn–Rokitnicki–Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases.

The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies.

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. Targeting of Tsg101 by RNA interference caused a strong reduction in Z-mediated budding. These results indicate that Z is the arenavirus functional counterpart of the matrix proteins found in other negative strand enveloped RNA viruses. Moreover, members of the vacuolar protein sorting pathway appear to play an important role in arena-virus budding. These findings open possibilities for antiviral strategies to combat LFV and other hemorrhagic fever arenaviruses.

Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein.

A recombinant S segment RNA (Sr) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) where the glycoprotein of vesicular stomatitis virus (VSVG) was substituted for the glycoprotein of LCMV (LCMV-GP) was produced intracellularly from cDNA under the control of a polymerase I promoter. Coexpression of the LCMV proteins NP and L allowed expression of VSVG from Sr. Infection of transfected cells with WT LCMV (LCMVwt) resulted in reassortment of the L segment of LCMVwt with the Sr at low frequency. Isolation of recombinant LCMV (rLCMV) expressing VSVG (rLCMV/VSVG) was achieved by selection against LCMVwt by using a cell line deficient in the cellular protease S1P. This approach was based on the finding that processing of LCMV-GP by S1P was required for virus infectivity. Characterization of protein and RNA expression of rLCMV/VSVG in infected cells confirmed the expected virus genome organization. rLCMV/VSVG caused syncytium formation in cultured cells and grew to approximately 100-fold lower titers than WT virus but, like the parent virus, it persisted in neonatally infected mice without clinical signs of disease.

Role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and RNA replication.

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.

Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV)

Passage of the prototypic arenavirus lymphocytic choriomenigitis virus (LCMV) in cultured cells in the presence of the mutagenic agent 5-fluorouracil (FU) resulted in efficient and systematic virus extinction under conditions that did not significantly affect cell survival. FU-mediated extinction of LCMV was associated with 3.6- to 10-fold increases in the mutation frequencies for the three viral genes examined, but with only very modest effects on virus replication and transcription during a single round of infection. Likewise, FU did not affect expression of a LCMV minigenome. In contrast, the well documented antiviral effect of ribavirin against LCMV was not associated with significant increases in virus mutation frequencies, but rather with a dramatic inhibition of both viral RNA synthesis and LCMV minigenome expression. Mutagen induced viral extinction has been recently reported for positive strand RNA viruses polio and foot-and-mouth disease, and the lentivirus HIV-1. Our findings indicate that lethal mutagenesis can be effective also against LCMV, a negative strand RNA virus. Moreover, FU treatment prevented the establishment of LCMV persistent infection in mice deficient in B and T cells, suggesting the feasibility in vivo of lethal mutagenesis as a novel antiviral strategy.

Characterization of the Genomic Promoter of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

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.

Neonates mount robust and protective adult-like CD8(+)-T-cell responses to DNA vaccines.

Neonates are thought to mount less vigorous adaptive immune responses than adults to antigens and infectious agents. This concept has led to a delay in the administration of many currently available vaccines until late infancy or early childhood. It has recently been shown that vaccines composed of plasmid DNA can induce both humoral and cell-mediated antimicrobial immunity when administered within hours of birth. In most of these studies, immune responses were measured weeks or months after the initial vaccination, and it is therefore questionable whether the observed responses were actually the result of priming of splenocytes within the neonatal period. Here we show that DNA vaccination at birth results in the rapid induction of antigen-specific CD8(+) T cells within neonatal life. Analyses of T-cell effector functions critical for the resolution of many viral infections revealed that neonatal and adult CD8(+) T cells produce similar arrays of cytokines. Furthermore, the avidities of neonatal and adult CD8(+) T cells for peptide and the rapidity with which they upregulate cytokine production after recall encounters with antigen are similar. Protective immunity against the arenavirus lymphocytic choriomeningitis virus, which is mediated by CD8(+) cytotoxic T cells, is also rapidly acquired within the neonatal period. Collectively these data imply that, at least in the case of CD8(+) T cells, neonates are not as immunodeficient as previously supposed and that DNA vaccines may be an effective and safe means of providing critical cell-mediated antiviral immunity extremely early in life.

Characterization of the arenavirus RING finger Z protein regions required for Z-mediated inhibition of viral RNA synthesis.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an enveloped virus with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides, namely, nucleoprotein; surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Using a reverse genetic system based on the ARM strain of LCMV, we have previously shown that Z has a strong inhibitory activity on LCMV minigenome transcription and RNA replication (T. I. Cornu and J. C. de la Torre, J. Virol. 75:9415-9426, 2001). In the present study, we have identified regions and specific amino acid residues within Z which contribute to its inhibitory activity on RNA synthesis mediated by the LCMV polymerase. Z proteins from different LCMV strains had similar inhibitory activities on the expression of the LCMV ARM minigenome, whereas the Z protein of the genetically more distantly related Tacaribe virus had an approximately 10-fold lower inhibitory activity on ARM minigenome expression. Results from the use of chimera proteins between Z and Xenopus Neuralized, a nonviral RING finger protein, indicated that the structural integrity of the Z RING domain (RD) was required but not sufficient for the inhibitory activity of Z. Serial deletion mutants of the N and C termini of Z showed that the N terminus (residues 1 through 16) and C terminus (residues 79 through 90) do not contribute to the Z inhibitory activity. A highly conserved tryptophan (W) residue located at position 36 in ARM-Z, next to the second conserved cysteine (C) residue of the Z RD, also contributed to the Z inhibitory activity.

Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles.

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral trans-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.