The primary structure of the lymphocytic choriomeningitis virus L gene encodes a putative RNA polymerase

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.Mutagenic agents administered alone or in combination with antiviral inhibitors can drive RNA virus populations to extinction (4, 18, 20, 21, 41, 42, 46, 48, 50, 51, 53, 65, 67, 72, 77, 79; reviewed in references 6, 27, 28, 31, and 39). Loss of infectivity has been interpreted as an irreversible transition that occurs when template copying fidelity falls below a critical value termed the error threshold. Such a transition has been termed virus entry into error catastrophe, or lethal mutagenesis (50), and its existence has been supported by several theoretical studies (5,31,60,83). In agreement with this concept, analysis of the mutant spectra of virus populations on their way to extinction has shown 2-to 17-fold increases in mutation frequencies, calculated among components of the mutant spectra, as well as increases in Shannon entropy (a measure of the different types of sequences present in a mutant spectrum) to nearly maximal values (that is, each component of the mutant spectrum showed a unique sequence) (reviewed in references 27 and 28).The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) (12,63,64) showed extreme sensitivity to 5-fluorouracil (FU)-induced mutagenesis (41, 72) compared with other RNA viruses subjected to comparable doses of the mutagen (79). The extreme sensitivity of LCMV to FU offered an opportunity to analyze the capacity of LCMV to regain infectivity following FU mutagenesis as well as the modification of genomic nucleotide sequence variations as the virus moves toward or away from the error threshold and to explore the possible dominance of hypermutated genomes in the transition to extinction. The results reveal a remarkable capacity of LCMV populations to modify their fitness level while maintaining an invariant consensus sequence. Multiple molecular clones were analyzed to define a sequence at nucleotides 470 to 550 within the intergenic region of genomic segment L. A number of standard and mutated oligonucleotide primers have failed to produce evidence of hypermutated viral sequences in the L polymerase gene. The results suggest that limited mutagenesis may be sufficient to ...

Section: Discussionmentioning

confidence: 77%

Mutagenesis-Induced, Large Fitness Variations with an Invariant Arenavirus Consensus Genomic Nucleotide Sequence

Grande-Pérez

Gómez-Mariano

Löwenstein

et al. 2005

“…The Z protein, produced from the large (L) genomic segment, is a small RING domain-containing matrix protein that mediates virus budding, regulates viral RNA synthesis, and mediates host immune suppression (4,5). The large L protein (ϳ200 kDa), also encoded on the L segment, is the RNA-dependent RNA polymerase (RdRp), which is required for viral RNA synthesis (6). The glycoprotein (GPC), encoded on the small (S) segment, is posttranslationally processed into a stable signal peptide (SSP), the receptor-binding G1 protein, and the transmembrane G2 protein (7).…”

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confidence: 99%

A Novel Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and Cellular Immunity after a Booster Dose

Dhanwani

Zhou

Huang

et al. 2016

Pichinde virus (PICV) is a bisegmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV (strain P18) with a trisegmented RNA genome (rP18tri), which encodes all four PICV gene products and as many as two foreign genes. We have engineered the vector to express the green fluorescent protein (GFP) reporter gene (abbreviated as G in virus designations) and either the hemagglutination (HA [H]) or the nucleoprotein (NP [P]) gene of the influenza A/PR8 virus. The trisegmented viruses rP18tri-G/H and rP18tri-G/P showed slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza virus challenge even 120 days after immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a primeand-boost vaccination strategy. IMPORTANCE We have developed a novel Pichinde virus (PICV)-based live viral vector, rP18tri, that packages three RNA segments and encodes as many as two foreign genes. Using the influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and can lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that distinguishes this from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.A renaviruses are enveloped RNA viruses with a bisegmented genome and mostly use rodents as natural hosts. There are at least 27 members that are geographically, serologically, and phylogenetically divided into Old World and New World arenaviruses (1). The prototypic lymphocytic choriomeningitis virus (LCMV) infection of mice has long been used as a valuable model with which to study viral persistence and virus-induced immunity and immunopathology (2, 3). The arenavirus is composed of a total of four genes on two genomic RNA segments in opposite orientations (1). The Z protein, produced from the large (L) genomic segment, is a small RING domain-containing matrix protein that mediates virus budding, regulates viral RNA synthesis, and mediates host immune suppression (4, 5). The large L protein (ϳ200 kDa), also encoded on the L segment, is the RNA-dependent RNA polymerase (RdRp), which is required for viral RNA synthesis (6). The glycoprotein (GPC), encoded...

“…Notably, none of the major changes observed were reversions to wild-type parental virus (MOPV or LASV) sequences, and none of CAA, or 5=-L segment-ACAAGTTGGAGGGAACAGGGACT), dNTPs, and Platinum Taq DNA polymerase High Fidelity (Invitrogen) in 50-l reaction mixtures. The amplified products (L segment, approximately 7.4 kb, and S segment, 3.4 kb) were separated in Tris-borate-EDTA (TBE)-agarose gels and purified using a QIAquick Gel Extraction Kit (Qiagen), and each DNA strand was sequenced using a primer-walking method (1,16,17). All sequencing reactions were performed in the Applied Biosystems 3130xl automated sequencer using BigDye terminators (Applied Biosystems), and the sequences were edited and assembled using Sequencher v4.6 (Gene Codes Corporation, Ann Arbor, MI).…”

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confidence: 99%

Genetic Variation In Vitro and In Vivo of an Attenuated Lassa Vaccine Candidate

Zapata

Goicochea

Nadai

et al. 2014

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.