Bisegmented Genome Research Articles

Molecular Mechanism of Arenavirus Assembly and Budding

Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2.

Picobirnaviruses in the Human Respiratory Tract

TO THE EDITOR: Picobirnaviruses (family Picobirnaviridae) are nonenveloped, double-stranded RNA viruses of vertebrates with a bisegmented genome. Segment 1 (2.2-2.7 kb) encodes the capsid protein, and segment 2 (1.2-1.9 kb) encodes the RNA-dependent RNA polymerase. On the basis of sequence diversity in segment 2, picobirnaviruses are classified into 2 genogroups (1-4). Picobirnaviruses have been detected in fecal samples from humans with and without gastroenteritis; in patients co-infected with known enteric pathogens, including rotaviruses, caliciviruses, and astroviruses (1,4); and in a wide range of animals, such as pigs, calves, dogs, monkeys, and snakes. The pathogenicity of picobirnaviruses largely remains to be determined, but studies in immunocompromised persons suggest that picobirnaviruses may be opportunistic enteric pathogens (5,6).

Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth

Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3' terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.

Identification of Two Functional Domains within the Arenavirus Nucleoprotein

Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L), and a RING finger matrix (Z) protein. The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (J. C. Casabona et al., J. Virol. 83:7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N-coding sequence, and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that N protein displays two functional domains. By using coimmunoprecipitation studies, VLP incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461 to 489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92 to 119) as a newly recognized molecular determinant of N homotypic interactions.

A novel putative partitivirus of the saprotrophic fungus Heterobasidion ecrustosum infects pathogenic species of the Heterobasidion annosum complex

We characterized the bisegmented genome of a putative double-stranded (ds) RNA virus from a Chinese isolate of the fungus Heterobasidion ecrustosum, a member of the Heterobasidion insulare species complex. The larger genomic segment of 1885 bp encoded a putative RNA dependent RNA polymerase (RdRp, 585 aa), and the smaller one for a putative coat protein of 521 aa (1826 bp). Phylogenetic analyses suggest that this novel virus species, named as ‘ Heterobasidion RNA virus 3 from H. ecrustosum, strain 1’ (HetRV3-ec1), can be assigned to the family Partitiviridae, being most similar to the Helicobasidium mompa dsRNA mycovirus with RdRp amino acid similarity of 54 %. The similarity to known viruses of other Heterobasidion species was notably low (25–39 %). The virus could be experimentally transmitted to members of the Heterobasidion annosum complex: the European Heterobasidion abietinum and North American Heterobasidion occidentale, and the original host strain could be cured from the virus by thermal treatment. Microscopical observations showed that hyphae of H. ecrustosum anastomosed occasionally with H. abietinum and H. occidentale, and suggested a possible route for horizontal transmission between these sexually incompatible species. The virus infection seemed to cause variable effects on the growth rate of its fungal hosts, but the results were strongly dependent on fungal strain, growth medium and incubation temperature.

Lassa Fever: Another Infectious Menace

Nigeria is presently suffering from another Lassa fever epidemic. This was confirmed in the statement of the Minister of Health of the Federation in which he said, “There has been an upsurge in the reported cases of Lassa fever since the beginning of this year, especially in the Federal Capital Territory and its environs. Within two weeks, 12 cases with five deaths due to the disease were recorded. 25 contacts are confirmed by laboratoryinvestigations to have been infected, including 4 health staff working in the National Hospital, Abuja.”1 Lassa fever is an acute viral haemorrhagic fever first described in 1969 in the town of Lassa in Borno state, Nigeria.2 It isendemic in West African countries, and causes 300,000 cases annually with 5000 deaths.3 Lassa fever epidemics occur in Nigeria, Liberia, Sierra Leone, Guinea and the Central African Republic.4 Lassa virus, the agent of the disease is a member of the Arenaviridae family. The virus is pleomorphic with single-stranded and bisegmented RNA genome.3 Its primary host is Natal Multimammate Mouse (Mastomys natalensis). Transmission to man occurs via exposure to the rat excrement through respiratory or gastrointestinal tracts5, exposure of broken skin or mucus membrane to infected material, direct contact, sexually and transplacentally. The prevalence of antibodies to the virus is 8-22%9 in Sierra Leone, 4-55% in Guinea,12 and 21% in Nigeria.13 Thedisease is mild or asymptomatic in 80% of infected people, but 20% have a severe multisystemic disease. Clinical features are difficult to differentiate from that of other viral haemorrhagic fevers and common febrile illness such as Malaria, Typhoid fever and so on. Definitive diagnosis is by viral isolation, Antigen and Antibody detection and ReverseTranscriptase PCR. Treatment is with Ribavirin, an antiviral agent. No vaccine is currently available. Prevention is by keeping rats away from homes.

The Nucleoprotein of Lymphocytic Choriomeningitis Virus Facilitates Spread of Persistent Infection through Stabilization of the Keratin Network

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus containing a bisegmented single-stranded RNA genome with an ambisense coding strategy. MX is a noncytolytic LCMV strain with an in vitro host range restricted to only few cell lines. MX LCMV spreads via cell-cell contacts and causes persistent infection with high production of viral nucleoprotein (NP). Using a proteomic approach, we identified keratin 1 (K1), an intermediate filament network component, as a binding partner of the viral NP. The functional significance of this interaction has been examined by chemical disruption of the keratin network, resulting in a reduced spread of MX LCMV in HeLa cells. However, K1 disassembly was considerably lower in MX LCMV-infected cells than in noninfected counterparts, indicating that NP can stabilize the keratin network and thereby support the integrity of cytoskeleton. The presence of NP also resulted in increased formation of desmosomes and stronger cell-cell adhesion. Similar effects were observed in HeLa cells persistently infected with LCMV strain Armstrong. Our findings suggest that the keratin network is important for the intercellular transmission of persistent LCMV infection in epithelial cells and show that the virus can actively facilitate its own intercellular spread through the interaction between the viral NP and K1 and stimulation of cell-cell contacts.

Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest

Several arenaviruses cause hemorrhagic fever disease in humans for which no licensed vaccines are available and current therapeutic intervention is limited to the off-label use of the wide-spectrum antiviral ribavirin. However, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has proven to be a Rosetta stone for the investigation of virus-host interactions. Arenaviruses have a bisegmented negative-strand RNA genome. The S segment encodes for the virus nucleoprotein and glycoprotein, whereas the L segment encodes for the virus polymerase (L) and Z protein. The ability to generate recombinant LCMV (rLCMV) expressing additional foreign genes of interest would open novel avenues for the study of virus-host interactions and the development of novel vaccine strategies and high-throughput screens to identify antiarenaviral molecules. To this end, we have developed a trisegmented (1L + 2S) rLCMV-based approach (r3LCMV). Each of the two S segments in r3LCMV was altered to replace one of the viral genes by a gene of interest. All r3LCMVs examined expressing different reported genes were stable both genetically and phenotypically and exhibited wild-type growth properties in cultured cells. Reporter gene expression in r3LCMV-infected cells provided an accurate surrogate of levels of virus multiplication. Notably, some r3LCMVs displayed highly attenuated virulence in mice but induced protective immunity against a subsequent lethal challenge with wild-type LCMV, supporting the potential development of r3LCMV-based vaccines.

Infectious bursal disease virus is an icosahedral polyploid dsRNA virus

Viruses are a paradigm of the economy of genome resources, reflected in their multiplication strategy and for their own structure. Although there is enormous structural diversity, the viral genome is always enclosed within a proteinaceous coat, and most virus species are haploid; the only exception to this rule are the highly pleomorphic enveloped viruses. We performed an in-depth characterization of infectious bursal disease virus (IBDV), a non-enveloped icosahedral dsRNA virus with a bisegmented genome. Up to 6 natural populations can be purified, which share a similar protein composition but show higher sedimentation coefficients as particle density increases. Stoichiometry analysis of their genome indicated that these biophysical differences correlate with the copy number of dsRNA segments inside the viral capsid. This is a demonstration of a functional polyploid icosahedral dsRNA virus. We show that IBDV particles with greater genome copy number have higher infectivity rates. Our results show an unprecedented replicative strategy for dsRNA viruses and suggest that birnaviruses are living viral entities encompassing numerous functional and structural characteristics of positive and negative ssRNA viruses.

Natural occurrence of a partitivirus in the sapstaining fungus Ceratocystis resinifera

The bisegmented genome of a double-stranded RNA (dsRNA) virus from isolate LG115 of Ceratocystis resinifera, a sapstaining ascomycete fungus, was characterized. The larger segment (dsRNA-1) was 2305 base pairs in length, whereas the smaller one (dsRNA-2) was 2207 base pairs. The positive strand of dsRNA-1 contained an open reading frame (ORF) with the potential to encode a protein of 661 amino acids, and this ORF was closely related to coat proteins (CPs) of previously characterized fungal partitiviruses. The dsRNA-2 sequence encodes a putative protein of 663 amino acids, and this protein contains conserved motifs of RNA-dependent RNA polymerases (RDRPs) and is highly related to RDRPs of fungal partitiviruses. These results suggest that these two dsRNAs are the genome of a partitivirus. Nucleotide and amino acid comparisons revealed high similarities between this virus and a partitivirus described from Ceratocystis polonica, a closely related sapstaining fungus. Over the entire genome, the two viruses shared 82.8%–84.7% nucleotide sequence identities, and the amino acid sequence identities for CPs and RDRPs were 87.3% and 95.6%, respectively. These results suggest that the viruses in C. polonica and C. resinifera can be considered as two strains of the same partitivirus. The natural occurrence of a partitivirus in two fungal species indicates that horizontal transmission of this partitivirus may have occurred between these two fungi. Northern blot analysis indicated that this partitivirus was widespread in populations of C. resinifera.

Detection of Genogroup I and II human picobirnaviruses showing small genomic RNA profile causing acute watery diarrhoea among children in Kolkata, India

Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55 kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children ( n = 20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus ( n = 3) or astrovirus ( n = 3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites ( Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria ( Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. ( n = 1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3–2.6 and 1.5–1.9 kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25–PicoB43 for Genogroup I and PicoB23–PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs ( n = 6) and Genogroup II PBVs ( n = 5), while some could not be amplified ( n = 3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries.

Lassa Virus Genome

Lassa virus (LASV), the most dangerous human pathogen among the Arenaviridae, belongs to a complex of genetically related virus strains responsible for the deaths of thousands of people in West Africa each year. The virus has a bi-segmented (L and S) single-stranded RNA genome. Each segment contains two genes in ambisense orientation. The L RNA encodes a large protein, L, or RdRp and a small zinc-binding, Z protein. The S RNA encodes the major structural proteins, nucleoprotein (NP) and glycoprotein precursor (GPC), cleaved into signal peptide, GP1, and GP2 glycoproteins. Genetic diversity among LASV strains is the highest within the family Arenaviridae and NP and RdRp genes are the most variable among LASV genes. The LASV genetic diversity is a great challenge for vaccine development. In addition to LASV and the prototype lymphocytic choriomeningitis virus (LCMV), the Old World group of arenaviruses includes three other related viruses, Mopeia (MOPV), Mobala (MOBV), and Ippy (IPPYV). These viruses as well as a MOP/LAS reassortant carrying the L RNA segment from MOPV and S RNA segment from LASV are non-pathogenic for experimental animals and are able to induce protective immunity against LASV. Lassa Fever pathogenesis is a sum of the effects induced by viral replication and immune response. The goal of this review is to cover recent publications on viral and host genes that control LASV virulence. The full-length genome sequence of LASV isolates and LASV-related nonpathogenic arenaviruses will provide a useful genetic tool to map LASV genes involved in virulence and to gain insight into phylogeny and evolution of the Old World arenaviruses. Keywords: Arenaviruses, Lassa fever, Lassa virus, RNA segments, genome

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.

Complementarity in the Supramolecular Design of Arenaviruses and Retroviruses Revealed by Electron Cryomicroscopy and Image Analysis

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from approximately 400 to approximately 2,000 A. The surface spikes were spaced approximately 100 A apart and extended approximately 90 A from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially approximately 30 and approximately 60 A from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of approximately 57 and approximately 74 A. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.

Cells expressing the RING finger Z protein are resistant to arenavirus infection.

Arenaviruses include Lassa fever virus (LFV) and the South American hemorrhagic fever viruses. These viruses cause severe human disease, and they pose a threat as agents of bioterrorism. Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides: nucleoprotein (NP); surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Our investigators have previously shown that Z has a strong inhibitory activity on RNA synthesis mediated by the polymerase of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). In this report we show that cells transduced with a replication-deficient recombinant adenovirus expressing Z (rAd-Z) are resistant to LCMV and LFV infection. Virus cell entry mediated by LCMV or LFV GP was not affected in rAd-Z-transduced cells, but both virus transcription and replication were strongly and specifically inhibited, which resulted in a dramatic reduction in production of infectious virus. These findings open new avenues for developing antiviral strategies to combat the highly pathogenic human arenaviruses, including LFV.

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces

To determine the incidence of rotavirus infection among dairy herds in the State of São Paulo, Brazil, 576 faecal samples obtained from calves aged 1–45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil.

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.

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.

A simple method for screening bacterial colonies for mutagenized sites in plasmid DNA

Because of the multiple-step process that is involved in the detection of mutagenized restriction enzyme sites in plasmid DNA, a simple and accurate method was developed to analyse the plasmid DNA of site-directed mutagenesis experiments from bacterial colonies. The desired mutated part is located between the Eco RI restriction site on pUC19. Two mutagenic primers were designed to replace only one nucleotide on segments A and B of the bi-segmented genome of infectious bursal disease virus (IBDV). Two restriction sites were created for those mutations in each segment, Fsp I and Dra I, respectively. Following a protocol from the site-directed mutagenesis kit, the mutated plasmids were used to transform, and were propagated and maintained in DH5 alpha competent cells. Colonies were picked from the master plate, and used as DNA template for PCR. The PCR technique included the design of two pairs of primers, one for each segment, which were to amplify a region up to 1000 bp. Samples were pre-incubated for 3 min at 94 °C to induce bacterial lysis before starting the nucleic acid amplification. The PCR products 918 bp from segment A and 650 bp from segment B were digested with Fsp I and Dra I at 37 °C for 1 h. Products were resolved on 0.9% agarose gel which contained ethidium bromide. This method is simpler, faster and more accurate than the traditional method of mini-prep plasmid isolation and colony blot hybridization to identify the mutated plasmids.

RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome.

Arenaviruses have a bisegmented negative-strand RNA genome whose proteomic capability is limited to only four polypeptides, namely, nucleoprotein (NP), surface glycoprotein (GP) that is proteolytically processed into GP1+GP2, polymerase (L), and a small (11-kDa) RING finger protein (Z). The role of Z during the Lymphocytic choriomeningitis virus (LCMV) life cycle is poorly understood. We investigated the function of Z in virus transcription and replication by using a reverse genetic system for the prototypic arenavirus LCMV. This system involves an LCMV minigenome and the minimal viral trans-acting factors (NP and L), expressed from separated cotransfected plasmids. Cotransfection of the Z cDNA strongly inhibited LCMV minigenome expression. The effect required synthesis of Z protein; its magnitude was dose dependent and occurred with levels of Z protein substantially lower than those observed in LCMV-infected cells. Coexpression of Z did not prevent the encapsidation of plasmid supplied minigenome, but it affected both transcription and RNA replication similarly. Mutations in Z that unfolded its RING finger domain eliminated its inhibitory activity, but RING proteins not related to Z did not affect LCMV minigenome expression. Consistent with the minigenome results, cells transiently expressing Z exhibited decreased susceptibility to infection with LCMV.