Julie K. Pfeiffer

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirins antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

Intestinal microbiota promote enteric virus replication and systemic pathogenesis

Commensal microflora promote the pathogenesis of mucosally acquired viruses. Intestinal bacteria aid host health and limit bacterial pathogen colonization. However, the influence of bacteria on enteric viruses is largely unknown. We depleted the intestinal microbiota of mice with antibiotics before inoculation with poliovirus, an enteric virus. Antibiotic-treated mice were less susceptible to poliovirus disease and supported minimal viral replication in the intestine. Exposure to bacteria or their N-acetylglucosamine–containing surface polysaccharides, including lipopolysaccharide and peptidoglycan, enhanced poliovirus infectivity. We found that poliovirus binds lipopolysaccharide, and exposure of poliovirus to bacteria enhanced host cell association and infection. The pathogenesis of reovirus, an unrelated enteric virus, also was more severe in the presence of intestinal microbes. These results suggest that antibiotic-mediated microbiota depletion diminishes enteric virus infection and that enteric viruses exploit intestinal microbes for replication and transmission.

Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice

RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.

Ribavirin Improves Early Responses to Peginterferon Through Improved Interferon Signaling

BACKGROUND & AIMS The therapeutic mechanisms of ribavirin for hepatitis C are unclear. Microarray analyses have shown that ribavirin increases induction of interferon-stimulated genes. We evaluated viral kinetics, serum cytokine expression, and viral mutagenesis during early stages of peginterferon therapy with and without ribavirin. METHODS Fifty patients with chronic hepatitis C virus (HCV) infection genotype 1 were randomly assigned to groups that were given peginterferon alpha-2a, with or without ribavirin, for 4 weeks; all patients then received an additional 44 weeks of combination therapy. First- and second-phase viral kinetics were evaluated. Serum levels of interferon-gamma-inducible protein-10 (IP10), monokine induced by interferon-gamma, and monocyte chemoattractant protein 1 were quantified as measures of the interferon-stimulated genes response. NS5A and NS5B were partially sequenced, and mutation rates were calculated. RESULTS The first-phase decrease in HCV RNA was similar between groups. Patients who received ribavirin had a more rapid second-phase decrease, compared with patients who did not receive ribavirin-particularly those with an adequate first-phase decrease (0.61 vs 0.35 log10 IU/mL/week; P = .018). At 12 hours, fold induction of serum IP10 was higher in patients given the combination therapy than those given peginterferon only (7.6- vs 3.8-fold; P = .01); however, the difference was greatest in patients with an adequate first-phase decrease in HCV RNA. IP10-induction correlated with first- and second-phase kinetics and with ribavirin serum concentrations on day 3. HCV mutation rates were similar between groups. CONCLUSIONS Ribavirin improves the kinetics of the early response to therapy in patients with an adequate initial response to peginterferon. Induction of interferon-stimulated cytokines correlates with viral kinetics following ribavirin therapy, suggesting that ribavirin promotes interferon signaling.

Bacterial Lipopolysaccharide Binding Enhances Virion Stability and Promotes Environmental Fitness of an Enteric Virus

Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication, and pathogenesis in mice are equivalent, VP1-T99K poliovirus was unstable in feces following peroral inoculation of mice. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host.

Ribavirin Resistance in Hepatitis C Virus Replicon-Containing Cell Lines Conferred by Changes in the Cell Line or Mutations in the Replicon RNA

ABSTRACT Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [3H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.

Transkingdom control of viral infection and immunity in the mammalian intestine

Microbial villages shape viral infections Viruses infecting the intestinal tract, such as noroviruses and rotaviruses, are major human pathogens. Despite facing an extreme environment within their hosts, which includes pH gradients, digestive enzymes, and the billions of microbes that inhabit human guts, these viruses somehow manage to survive and often thrive. Pfeiffer and Virgin review the complex microbial encounters that occur between enteric viruses and gut microbiota. Trans-kingdom interactions (that is, between viruses, bacteria, archaea, helminthes, fungi, and phage) are particularly important in shaping the course of a viral infection and the ensuing host immune response. Science, this issue p. 10.1126/science.aad5872 BACKGROUND Viruses that infect the mammalian gastrointestinal tract have intimate relationships with the host, as well as members of the complex community of microbes that inhabit the intestine. The mammalian intestine contains the highest density of microbes in the body. These microbes, collectively referred to as the intestinal microbiota, include bacteria, archaea, fungi, viruses, and eukaryotes. Emerging data indicate that enteric viruses regulate, and are regulated by, these other microbes through a series of processes termed “transkingdom interactions.” Recent advances have shed light on the nature and importance of these transkingdom interactions for enteric virus replication, transmission, and disease. ADVANCES The study of enteric virus pathogenesis has traditionally focused on viral virulence genes of classical pathogens and host immunity to these agents. Recent analysis of the viruses present in the intestine has revealed a dynamic and diverse taxonomic intestinal viral world (the enteric virome) featuring, in addition to recognized enteric viral pathogens, many new viruses and new relationships between known viral types and disease. We now know that enteric viral infection must be considered in light of the fact that viruses are part of a complex milieu of microbes and microbial products that directly and indirectly regulate viral pathogenesis. Thus, interactions of enteric viruses with other microbes are increasingly recognized as critical to viral infectivity, disease, and control. Studies leveraging the simple paradigm of examining enteric viruses in the intestine after natural oral infection have driven the field forward. It is now clear that members of the intestinal microbiome promote replication and transmission of enteric viruses from four different families: noroviruses, picornaviruses, retroviruses, and reoviruses. Therefore, the standard reductionist approach of understanding the pathogenesis of, and immunity to, viral infection in the context of a single virus interacting with a single host is too limited to capture the full range of relevant pathogenic mechanisms. This simple concept has broad implications for prevention and therapy of viral infections of great medical importance. OUTLOOK Despite recent rapid advances, there are still major gaps in our understanding of transkingdom control of enteric virus replication, pathogenesis, and transmission. Recognition of the important impact of the bacterial microbiota has advanced more rapidly than for any other component of the intestinal microbiota. A particular challenge for studies to comprehensively identify viruses within the enteric virome is their diversity and extreme sequence variability. A key direction for the field is to identify functional relationships governing transkingdom interactions in the intestine, including the dynamic coevolved relationship between the intestinal microbiota and innate and adaptive immunity. The field is now poised to define, in structural and biochemical terms, specific molecular mechanisms responsible for such transkingdom interactions. Future studies on the mammalian virome and transkingdom factors that influence viral infection may inspire new therapeutic approaches. In this Review, we explore the interplay between viruses, the microbiota, and host immune responses. We highlight how transkingdom interactions influence infection with mammalian enteric viruses, including pathogens. Intestinal microbiota promote enteric virus replication. Enteric viruses can interact with bacteria before initiating replication in the mammalian intestine. This illustration shows norovirus interacting with bacteria. Through these interactions, and/or through microbiota-mediated alteration of host immune responses, intestinal microbiota facilitate enteric virus replication in the gut. [Credit: K. Sutliff/Science] Viruses that infect the intestine include major human pathogens (retroviruses, noroviruses, rotaviruses, astroviruses, picornaviruses, adenoviruses, herpesviruses) that constitute a serious public health problem worldwide. These viral pathogens are members of a large, complex viral community inhabiting the intestine termed “the enteric virome.” Enteric viruses have intimate functional and genetic relationships with both the host and other microbial constituents that inhabit the intestine, such as the bacterial microbiota, their associated phages, helminthes, and fungi, which together constitute the microbiome. Emerging data indicate that enteric viruses regulate, and are in turn regulated by, these other microbes through a series of processes termed “transkingdom interactions.” This represents a changing paradigm in intestinal immunity to viral infection. Here we review recent advances in the field and propose new ways in which to conceptualize this important area.

Multiple Host Barriers Restrict Poliovirus Trafficking in Mice

RNA viruses such as poliovirus have high mutation rates, and a diverse viral population is likely required for full virulence. We previously identified limitations on poliovirus spread after peripheral injection of mice expressing the human poliovirus receptor (PVR), and we hypothesized that the host interferon response may contribute to the viral bottlenecks. Here, we examined poliovirus population bottlenecks in PVR mice and in PVR mice that lack the interferon α/β receptor (PVR-IFNAR−/−), an important component of innate immunity. To monitor population dynamics, we developed a pool of ten marked polioviruses discriminated by a novel hybridization-based assay. Following intramuscular or intraperitoneal injection of the ten-virus pool, a major bottleneck was observed during transit to the brain in PVR mice, but was absent in PVR-IFNAR−/− mice, suggesting that the interferon response was a determinant of the peripheral site-to-brain bottleneck. Since poliovirus infects humans by the fecal–oral route, we tested whether bottlenecks exist after oral inoculation of PVR-IFNAR−/− mice. Despite the lack of a bottleneck following peripheral injection of PVR-IFNAR−/− mice, we identified major bottlenecks in orally inoculated animals, suggesting physical barriers may contribute to the oral bottlenecks. Interestingly, two of the three major bottlenecks we identified were partially overcome by pre-treating mice with dextran sulfate sodium, which damages the colonic epithelium. Overall, we found that viral trafficking from the gut to other body sites, including the CNS, is a very dynamic, stochastic process. We propose that multiple host barriers and the resulting limited poliovirus population diversity may help explain the rare occurrence of viral CNS invasion and paralytic poliomyelitis. These natural host barriers are likely to play a role in limiting the spread of many microbes.

Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance

ABSTRACT Treatment for hepatitis C virus infection currently consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. Although RBV clearly plays a role in aiding the treatment response, its antiviral mechanism is unclear. Regardless of the specific mechanism of RBV, we hypothesize that differences in levels of cellular uptake of RBV may affect antiviral efficacy and treatment success and that cells may become RBV resistant through reduced uptake. We monitored RBV uptake in various cell lines and determined the effect of uptake capacity on viral replication. RBV-resistant cells demonstrated reduced RBV uptake and increased growth of a model RNA virus, poliovirus, in the presence of RBV. Overexpression of equilibrative nucleoside transporter 1 (ENT1) or concentrative nucleoside transporter 3 (CNT3) increased RBV uptake in RBV-sensitive cell lines and restored the uptake defect in most RBV-resistant cell lines. However, CNT3 is not expressed in Huh-7 liver cells, and inhibition of concentrative transport did not affect RBV uptake. Blocking equilibrative transport using the inhibitor nitrobenzylmercaptopurine riboside recapitulated the RBV-resistant phenotype in RBV-sensitive cell lines, with a reduction in RBV uptake and increased poliovirus growth. Taken together, these results indicate that RBV uptake is restricted primarily to ENT1 in the cell lines examined. Interestingly, some RBV-resistant cell lines may compensate for reduced ENT1-mediated nucleoside uptake by increasing the activity of an alternative nucleoside transporter, ENT2. It is possible that RBV uptake affects the antiviral treatment response, either through natural differences in patients or through acquired resistance.

Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages

ABSTRACT It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral “headful” was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19.2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.