An increase in the mutation rate during replication of RNA viruses can result in a decrease of viral infectivity and occasional virus extinction (11,34,39,40,60). Studies with the important animal pathogen foot-and-mouth disease virus (FMDV)-a member of the Picornaviridae family (53, 63)-have shown that a small replicative population size and low viral fitness favored virus extinction (60). This was documented with single and multiple passages of FMDVs differing up to 10 6 -fold in relative fitness in the absence or presence of the mutagenic base analogs 5-fluorourocil (FU) or 5-azacytidine, individually or in combination (59, 60). Mutagenic treatments resulted in occasional, not systematic, viral extinction, while parallel passages in the absence of mutagens never led to loss of infectivity, no matter how low the initial viral population size and fitness were (59, 60). These results suggested the possibility that a combination of an antiviral inhibitor, to reduce the replicative load of virus, and a mutagenic agent could be more effective in producing viral extinction than a mutagenic agent used in isolation. To test this possibility we have studied the effect of the mutagenic base analog FU and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of FMDV clones and populations depicting a wide range of relative fitness values. FU has been shown to be mutagenic for a number of RNA viruses (6,20,31,34,51,71), including FMDV (59,60). G at millimolar concentrations blocks the replication of picornaviruses (5,7,15,49,52,55), arboviruses (27), and several plant viruses (13, 67). In poliovirus, the target of G is the ATPase activity of nonstructural protein 2C (49), a protein involved in viral replication and encapsidation. In FMDV, amino acid substitutions at 2C have also been associated with resistance to G (56). Heparins are sulfated polysaccharides (9) which bind FMDV when the virus has been passaged in cell culture and has adapted to using heparan sulfate (HS) as a receptor (2,35,54). Adaptation to use of HS as a receptor has been associated with substitutions which lead to positively charged amino acids at exposed positions of the capsid (2, 54).Here we report that high-frequency extinctions of FMDV of low and intermediate fitness values can be achieved with a combination of FU and G but not with either drug alone. Extinction of high-fitness FMDV populations required a triple combination of G and H together with FU. Mutation frequencies in the mutant spectrum of three genomic regions of a preextinction population obtained by the combined action of an inhibitor and a mutagen were compared to values in genomes passaged in standard conditions and also to values previously determined for FMDV populations subjected to one or multiple passages in the presence of FU (60). We found that mutation frequencies increased up to fourfold. There was also a statistically significant increase in the number of mutations in preextinction populations with respect to control populations, and the ...

BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) can be cured of virus by treatment with the antiviral nucleoside analogue ribavirin. To study whether the process involved an increase in the number of mutations in the mutant spectrum of the viral population, viral genomes were cloned from persistently infected cells treated or untreated with ribavirin. An increase of up to 10-fold in mutation frequencies associated with ribavirin treatment was observed in the viral genomes from the treated cultures as compared with parallel, untreated cultures. To address the possible mechanisms of enhanced mutagenesis, we investigated the mutagenic effects of ribavirin together with guanosine, and mycophenolic acid in the presence or absence of guanosine. Changes in the intracellular nucleotide concentrations were determined for all treatments. The results suggest that the increased mutation frequencies were not dependent on nucleotide pool imbalances or due to selection of preexisting genomes but they were produced by a mutagenic action of ribavirin.

RNA viruses replicate near the error threshold for maintenance of genetic information, and an increase in mutation frequency during replication may drive RNA viruses to extinction in a process termed lethal mutagenesis. This report addresses the efficiency of extinction (versus escape from extinction) of foot-andmouth disease virus (FMDV) by combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H). Selection of G-or H-resistant, extinctionescape mutants occurred with low-fitness virus only in the absence of FU and with high-fitness virus with some mutagen-inhibitor combinations tested. The combination of FU, G, and H prevented selection of extinctionescape mutants in all cases examined, and extinction of high-fitness FMDV could not be achieved by equivalent inhibitory activity exerted by the nonmutagenic agents. The G-resistant phenotype was mapped in nonstructural protein 2C by introducing the relevant mutations in infectious cDNA clones. Decreases in FMDV infectivity were accompanied by modest decreases in the intracellular and extracellular levels of FMDV RNA, maximal intracellular concentrations of FU triphosphate, and a decrease in the intracellular concentrations of UTP. In addition to indicating a key participation of mutagenesis in virus extinction, the results suggest that picornaviruses provide versatile experimental systems to approach the problem of extinction failure associated with inhibitor-escape mutants during treatments based on enhanced mutagenesis.The error rate during template copying by viral RNA-dependent RNA polymerases and reverse transcriptases is close to the maximum tolerable for the maintenance of the genetic information of the virus. This is supported by theoretical predictions (reviewed in references 14 and 15) and experimental results on the adverse effects of enhanced mutagenesis on infectivity during cytolytic or persistent infections of viruses as diverse as picornaviruses (1, 9, 10, 23, 35, 52), retroviruses and retroviral vectors (29,30,37), the rhabdovirus vesicular stomatitis virus (VSV) (23), the flavivirus GB virus B (27), the arenavirus lymphocytic choriomeningitis virus (LCMV) (21, 46), and Hantaan virus (50).Virus extinction by enhanced mutagenesis was accompanied by increases in mutant spectrum complexity, as quantitated by mutation frequency and Shannon entropy (1,21,35,46,52). LCMV showed a very high sensitivity to extinction by 5-fluorouracil (FU) during persistent infections in BHK-21 cells (21, 46), and recently it has been shown that administration of FU to mice prevented the establishment of a persistent LCMV infection (46), providing the first evidence of feasibility of a lethal mutagenesis approach to control viral infections in vivo.Studies with the important animal pathogen foot-and-mouth disease virus (FMDV) have documented that low viral relative fitness and low viral load favored FMDV extinction by enhanced mutagenesis (35,52). The effect of fitness was evidenced using FMDV clo...

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.High mutation rates and quasispecies dynamics (17,21,23) confer great adaptability to RNA viruses and represent a major obstacle for the prevention and control of RNA viral diseases (17, 25). However, theoretical studies have provided evidence that for any replication system there is a maximum error rate compatible with maintenance of the information encoded in the replicating genome (2,22,23,37,46). The larger the complexity of the genome, the higher the copying fidelity needed to maintain the encoded information. The irreversible transition into loss of genetic information is termed entry into error catastrophe, and the critical average error rate at the transition point is the error threshold (2,22,23,37,46). This concept has encouraged research in a new antiviral strategy termed lethal mutagenesis (12,22,29,32). In the case of viruses, crossing the error threshold should result in a transition from a productive to an abortive infection. Virus extinction associated with enhanced mutagenesis has been documented with a variety of virus-host systems (1, 12, 13, 27, 29, 31-34, 39, 43, 44; for reviews, see references 16, 22, and 26), including prevention of the establishment of a persistent lymphocytic choriomeningitis virus (LCMV) infection in vivo (42).Studies with the animal picornavirus foot-and-mouth disease virus (FMDV) in cell culture have established that virus extinction through enhanced mutagenesis is favored by low viral load and low viral fitness (44). As a consequence, combination treatments involving a mutagenic agent and antiviral inhibitors were more effective than a mutagenic agent alone in driving FMDV to extinction (39). When the mutagenic activity was insufficient for a given fitness level of FMDV, inhibitor-resistant, e...

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Receptor classCellular structure a Extracellular matrix components, sugar derivatives and lipids GalactosylceramideGangliosides Glycosaminoglycans (heparan and chondroitin sulfates) Phospholipids Sialic acid (N-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid and N-glycolylneuraminic acid 3-O-sulfated heparan sulfate Cell adhesion and cell-cell contact proteins -Dystroglycan Coxsackievirus-adenovirus receptor (CAR; Ig superfamily) CD4 (Ig superfamily) CEACAMs (including Bgp1a, Bgp2, and pregnancy-specific glycoprotein) Intercellular adhesion molecule type 1 (ICAM-1; Ig superfamily) Integrins Junction adhesion molecule (JAM) Laminin receptor (high affinity) MHC class I and 2 -microglobulin Neural cell adhesion molecule (NCAM) Signaling lymphocyte activation molecule

The nucleocapsid protein VP35 of Marburgvirus, a filovirus, acts as the cofactor of the viral polymerase and plays an essential role in transcription and replication of the viral RNA. VP35 forms complexes with the genome encapsidating protein NP and with the RNA-dependent RNA polymerase L. In addition, a trimeric complex had been detected in which VP35 bridges L and the nucleoprotein NP. It has been presumed that the trimeric complex represents the active polymerase bound to the nucleocapsid. Here we present evidence that a predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homo-oligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (i) the probability to form coiled coils, (ii) homo-oligomerization, and (iii) the function of VP35 in viral RNA synthesis. Further, it was found that homo-oligomerization-negative mutants of VP35 could not bind to L. Thus, it is presumed that homo-oligomerization-negative mutants of VP35 are unable to recruit the polymerase to the NP/RNA template. In contrast, inability to homo-oligomerize did not abolish the recruitment of VP35 into inclusion bodies, which contain nucleocapsid-like structures formed by NP. Finally, transcriptionally inactive mutants of VP35 containing the functional homo-oligomerization domain displayed a dominant-negative phenotype. Inhibition of VP35 oligomerization might therefore represent a suitable target for antiviral intervention.Marburgvirus (MARV) and the closely related Ebolavirus (EBOV) together make up the family Filoviridae, which is classified in the order Mononegavirales. MARV causes a fulminant hemorrhagic fever in humans and nonhuman primates with high fatality rates (28). To date, neither a vaccine nor a curative treatment for MARV infection in humans is available. However, live attenuated recombinant vaccines have been described which protected nonhuman primates against MARV and EBOV infections (23). These might represent promising candidate vaccines for human use as well. The recent outbreak of MARV disease in Angola underlines the emerging potential of this pathogen (4). The MARV particle is composed of seven structural proteins. Four of them, NP, VP35, VP30, and L, constitute the nucleocapsid complex of MARV, which surrounds the viral genome. NP, the major nucleocapsid protein, self-assembles into tubular nucleocapsid-like structures, which are found intracellularly in large inclusions (25,29). The formation of the tubular structures by NP is presumed to represent the first step in the assembly of the nucleocapsid. NP interacts with VP35, which in turn interacts with the RNAdependent RNA polymerase L (3). The complex of VP35 and L represents the active RNA-dependent RNA polymerase, with VP35 serving as a polymerase cofactor (33). Additionally, a trimeric complex was observed consisting of NP, VP35, and L, with VP35 connecting L and NP (3). Three of the four nucleocapsid proteins, NP, VP35, and L, are essential for transcription and replication of the viral ...

Targeted gene transduction to organs and tissues of interest is the ultimate goal of therapeutic gene delivery. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration into undesired cell types poses a serious safety concern for their use in the clinic. Here we report the development of a new dual-targeted LV that can preferentially home to and express in prostate cancer bone metastases in vivo after systemic delivery. Transductional targeting is mediated by a modified Sindbis virus envelope that interacts with the prostate stem cell antigen (PSCA) expressed by prostate cancer cells, and transcriptional targeting is mediated by a prostate cell specific promoter. Homing to prostate tumors was achieved in 70% of the animals. Importantly, tumors could be detected in some cases by molecular imaging prior to X-ray detection. The dual-targeted vector presents enhanced specificity with respect to individual transcriptional or transductional targeted vectors. Transgene expression in the liver was 190 times lower than the expression associated with solely transductionally targeted vectors, and there was 12 times less vector DNA than the amount present with solely transcriptionally targeted vectors. The LV presented here is a powerful tool for obtaining stable and site-specific gene expression and can be easily modified for its use in other diseases.