Mar Perez

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.

Myristoylation of the RING Finger Z Protein Is Essential for Arenavirus Budding

ABSTRACT 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 [3H]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 LCMV-infected 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.

Role of the Virus Nucleoprotein in the Regulation of Lymphocytic Choriomeningitis Virus Transcription and RNA Replication

ABSTRACT 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.

Identification of the Lymphocytic Choriomeningitis Virus (LCMV) Proteins Required To Rescue LCMV RNA Analogs into LCMV-Like Particles

ABSTRACT 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.

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

ABSTRACT 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.

Characterization of the interaction of lassa fever virus with its cellular receptor α-dystroglycan

ABSTRACT The cellular receptor for the Old World arenaviruses Lassa fever virus (LFV) and lymphocytic choriomeningitis virus (LCMV) has recently been identified as α-dystroglycan (α-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 α-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 α-DG for infection of cells. Mapping of the binding site of LFV on α-DG revealed that LFV binding required the same domains of α-DG that are involved in the binding of LCMV. Further, LFV was found to efficiently compete with laminin α1 and α2 chains for α-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.

Arenavirus Z-Glycoprotein Association Requires Z Myristoylation but Not Functional RING or Late Domains

ABSTRACT Generation of infectious arenavirus-like particles requires the virus RING finger Z protein and surface glycoprotein precursor (GPC) and the correct processing of GPC into GP1, GP2, and a stable signal peptide (SSP). Z is the driving force of arenavirus budding, whereas the GP complex (GPc), consisting of hetero-oligomers of SSP, GP1, and GP2, forms the viral envelope spikes that mediate receptor recognition and cell entry. Based on the roles played by Z and GP in the arenavirus life cycle, we hypothesized that Z and the GPc should interact in a manner required for virion formation. Here, using confocal microscopy and coimmunoprecipitation assays, we provide evidence for subcellular colocalization and biochemical interaction, respectively, of Z and the GPc. Our results from mutation-function analysis reveal that Z myristoylation, but not the Z late (L) or RING domain, is required for Z-GPc interaction. Moreover, Z interacted directly with SSP in the absence of other components of the GPc. We obtained similar results with Z and GPC from the prototypical arenavirus lymphocytic choriomeningitis virus and the hemorrhagic fever arenavirus Lassa fever virus.

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

ABSTRACT 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.

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 ≈100-fold lower titers than WT virus but, like the parent virus, it persisted in neonatally infected mice without clinical signs of disease.

Cellular Entry of Lymphocytic Choriomeningitis Virus

ABSTRACT 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 α-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.