Minireplicon System Research Articles

Twenty natural amino acid substitution screening at the last residue 121 of influenza A virus NS2 protein reveals the critical role of NS2 in promoting virus genome replication by coordinating with viral polymerase.

Both influenza A virus genome transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the influenza RNA polymerase (FluPol), are dynamically regulated across the virus life cycle. It has been reported that the last amino acid I121 of the viral NS2 protein plays a critical role in promoting viral genome replication in influenza mini-replicon systems. Here, we performed a 20 natural amino acid substitution screening at residue NS2-I121 in the context of virus infection. We found that the hydrophobicity of the residue 121 is essential for virus survival. Interestingly, through serial passage of the rescued mutant viruses, we further identified adaptive mutations PA-K19E and PB1-S713N on FluPol which could effectively compensate for the replication-promoting defect caused by NS2-I121 mutation in the both mini-replicon and virus infection systems. Structural analysis of different functional states of FluPol indicates that PA-K19E and PB1-S713N could stabilize the replicase conformation of FluPol. By using a cell-based NanoBiT complementary reporter assay, we further demonstrate that both wild-type NS2 and PA-K19E/PB1-S713N could enhance FluPol dimerization, which is necessary for genome replication. These results reveal the critical role NS2 plays in promoting viral genome replication by coordinating with FluPol.IMPORTANCEThe intrinsic mechanisms of influenza RNA polymerase (FluPol) in catalyzing viral genome transcription and replication have been largely resolved. However, the mechanisms of how transcription and replication are dynamically regulated remain elusive. We recently reported that the last amino acid of the viral NS2 protein plays a critical role in promoting viral genome replication in an influenza mini-replicon system. Here, we conducted a 20 amino acid substitution screening at the last residue 121 in virus rescue and serial passage. Our results demonstrate that the replication-promoting function of NS2 is important for virus survival and efficient multiplication. We further show evidence that NS2 and NS2-I121 adaptive mutations PA-K19E/PB1-S713N regulate virus genome replication by promoting FluPol dimerization. This work highlights the coordination between NS2 and FluPol in fulfilling efficient genome replication. It further advances our understanding of the regulation of viral RNA synthesis for influenza A virus.

A GFP-expressing minigenome of a chrysovirus replicating in fungi

The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum, the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi.

Characterization of a novel reporter system for beak and feather disease virus

Beak and feather disease virus (BFDV) belongs to the Circoviridae family, which has a relatively simple replication mechanism. As BFDV lacks a mature cell culture system, a novel mini-replicon system based on the reporter plasmid that contains the origin of replication, which can bind to the Rep protein expressed from another plasmid and thus trigger its replication and induce/increase luminescence was developed. The dual-luciferase assay was used in this system to measure replicative efficiency by comparing relative light units (RLU) of firefly luciferase. Linear relationships between the luciferase activity of the reporter plasmids with the BFDV origin of replication and the amounts of the Rep protein and vice versa were found, suggesting the mini-replicon system can be used to quantify viral replication. Moreover, the activities of reporter plasmids driven by mutated Rep proteins or the activities of reporter plasmids with mutations were significantly downregulated. The Rep and Cap promoter activities can be characterized using this luciferase reporter system. Notably, the RLU of the reporter plasmid was considerably inhibited in the presence of sodium orthovanadate (Na3VO4). When BFDV-infected birds were treated with Na3VO4, the viral loads of BFDV rapidly decreased. In conclusion, this mini-replicon reporter gene-based system provides a practical means to screen for anti-viral drug candidates.

Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants.

Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.

Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement.

Rice stripe virus (RSV), a tenuivirus with four negative-sense/ambisense genome segments, is one of the most devastating viral pathogens affecting rice production in many Asian countries. Despite extensive research, our understanding of RSV infection cycles and pathogenesis has been severely impaired by the lack of reverse genetics tools. In this study, we have engineered RSV minireplicon (MR)/minigenome cassettes with reporter genes substituted for the viral open reading frames in the negative-sense RNA1 or the ambisense RNA2-4 segments. After delivery to Nicotiana benthamiana leaves via agroinfiltration, MR reporter gene expression was detected only when the codon-optimized large viral RNA polymerase protein (L) was coexpressed with the nucleocapsid (N) protein. MR activity was also critically dependent on the coexpressed viral suppressors of RNA silencing, but ectopic expression of the RSV-encoded NS3 silencing suppressor drastically decreased reporter gene expression. We also developed intercellular movement-competent MR systems with the movement protein expressed either in cis from an RNA4-based MR or in trans from a binary plasmid. Finally, we generated multicomponent replicon systems by expressing the N and L proteins directly from complementary-sense RNA1 and RNA3 derivatives, which enhanced reporter gene expression, permitted autonomous replication and intercellular movement, and reduced the number of plasmids required for delivery. In summary, this work enables reverse genetics analyses of RSV replication, transcription, and cell-to-cell movement and provides a platform for engineering more complex recombinant systems.

Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants.

Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

The critical region for viral RNA encapsidation in leader promoter of Nipah virus.

Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43–55) and the remaining region (aa 56–295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses.

Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts.IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter.

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25-30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Identification of two essential aspartates for polymerase activity in parainfluenza virus L protein by a minireplicon system expressing secretory luciferase.

Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.

The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication.

Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member of the Microrchidia family that is associated with the nuclear matrix, during virus infection. NXP2/MORC3 associates with the viral ribonucleoproteins in infected cells. Downregulation of NXP2/MORC3 reduced virus titers and accumulations of viral genomic RNA and mRNAs. Silencing of NXP2/MORC3 in an influenza virus CAT minireplicon system diminished CAT protein and cat mRNA levels but not genomic RNA levels. We propose that NXP2/MORC3 plays a role in influenza virus transcription.

186: Role of GTP-mediated structural changes of MxA on its antiviral function against influenza A

Mx proteins are interferon type I induced effector proteins and members of the family of dynamin-like GTPases. Human MxA protein exerts antiviral activity against a broad range of viruses including influenza A. The GTPase activity of MxA is essential for its proper antiviral function. Based on extensive mutational biochemical analyses we proposed a model where Mx proteins can assemble into highly ordered oligomers but exert their antiviral activity as monomers. Moreover, we have shown that human MxA physically interacts with the cellular DExD/h-box RNA helicases UAP56 and URH49 that are required for efficient replication of influenza A virus. Recently Gao and coworkers determined the crystal structure of MxA, revealing a globular GTP-binding domain and a four helical bundle stalk domain. In vitro, MxA multimerizes into large ring-like structures, involving three interaction interfaces. Mutations in the interfaces of MxA interfere with the oligomer formation, but, depending on the mutation, some MxA variants are still able to form monomers, dimers or tetramers. To date it is still a matter of debate whether MxA exerts antiviral activity in form of monomers, dimers and/or higher oligomeric structures. In order to assess whether GTP-binding has an effect on the stoichiometry of MxA we purified recombinant wildtype MxA and various mutants thereof and assessed the number of MxA protomers in the presence or absence GTP-γS by multi angle laser light scattering (MALLS). Surprisingly, we observed that in the presence of GTP-γS wildtype MxA converted from oligomers to tetramers and dimers. Similarly, addition of GTP-γS to the tetrameric mutant MxA (R640A) resulted in its dimerization. Furthermore, dimeric and monomeric MxA variants did not alter their stoichiometry upon addition of GTP-γS. Further, we evaluated the antiviral activity of wild type MxA and MxA variants employing an influenza A minireplicon system and influenza A virus infection of MxA expressing cells. The data clearly indicated that certain tetrameric, dimeric or monomeric variants of MxA were still able to efficiently inhibit virus replication while MxA mutants deficient for GTP binding were inactive. We currently test whether binding of UAP56 by MxA is required for its antiviral activity.

Construction of a Sonchus Yellow Net Virus Minireplicon: a Step toward Reverse Genetic Analysis of Plant Negative-Strand RNA Viruses

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.

Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly

Mx1 is a GTPase that is part of the antiviral response induced by type I and type III interferons in the infected host. It inhibits influenza virus infection by blocking viral transcription and replication, but the molecular mechanism is not known. Polymerase basic protein 2 (PB2) and nucleoprotein (NP) were suggested to be the possible target of Mx1, but a direct interaction between Mx1 and any of the viral proteins has not been reported. We investigated the interplay between Mx1, NP, and PB2 to identify the mechanism of Mx1's antiviral activity. We found that Mx1 inhibits the PB2-NP interaction, and the strength of this inhibition correlated with a decrease in viral polymerase activity. Inhibition of the PB2-NP interaction is an active process requiring enzymatically active Mx1. We also demonstrate that Mx1 interacts with the viral proteins NP and PB2, which indicates that Mx1 protein has a direct effect on the viral ribonucleoprotein complex. In a minireplicon system, avian-like NP from swine virus isolates was more sensitive to inhibition by murine Mx1 than NP from human influenza A virus isolates. Likewise, murine Mx1 displaced avian NP from the viral ribonucleoprotein complex more easily than human NP. The stronger resistance of the A/H1N1 pandemic 2009 virus against Mx1 also correlated with reduced inhibition of the PB2-NP interaction. Our findings support a model in which Mx1 interacts with the influenza ribonucleoprotein complex and interferes with its assembly by disturbing the PB2-NP interaction.

Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from East Africa, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function. LASV and MOPV L protein was also active in conjunction with LCMV NP, while the LCMV L protein required homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP (residues 340 to 558) as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. In conclusion, components of the replication complex of Old World arenaviruses have the potential to functionally and physically interact across species boundaries. Residue 53 and the C-terminal domain of NP are important for function of L protein during genome replication and transcription.

Domain Structure of Lassa Virus L Protein

The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures.

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

BackgroundChicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.Methodology/Principal FindingsChicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.Conclusions/SignificanceThe chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.

Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion

RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3' terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at approximately 60% of WT levels, and its product was found to be initiated at the WT position (-1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved.