Karla Kirkegaard

Subversion of Cellular Autophagosomal Machinery by RNA Viruses

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles.

ABSTRACT All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

Cellular autophagy: surrender, avoidance and subversion by microorganisms

Intracellular bacteria and viruses must survive the vigorous antimicrobial responses of their hosts to replicate successfully. The cellular process of autophagy — in which compartments bound by double membranes engulf portions of the cytosol and then mature to degrade their cytoplasmic contents — is likely to be one such host-cell response. Several lines of evidence show that both bacteria and viruses are vulnerable to autophagic destruction and that successful pathogens have evolved strategies to avoid autophagy, or to actively subvert its components, to promote their own replication. The molecular mechanisms of the avoidance and subversion of autophagy by microorganisms will be the subject of much future research, not only to study their roles in the replication of these microorganisms, but also because they will provide — as bacteria and viruses so often have — unique tools to study the cellular process itself.

The mechanism of RNA recombination in poliovirus.

Abstract We have investigated RNA recombination among poliovirus genomes by analyzing both intratypic and intertypic recombinant crosses involving the same defined genetic markers. Sequence analysis of the recombinant junctions of 13 nonsibling intertypic recombinants showed that intertypic RNA recombination is not site-specific, nor does it require extensive homology between the recombining parents at the crossover site. To discriminate between breaking-rejoining and copy choice mechanisms of RNA recombination, we have inhibited the replication of the recombining parents independently and found opposite effects on the frequency of genetic recombination in intratypic crosses. The results strongly support a copy choice mechanism for RNA recombination, in which the viral RNA polymerase switches templates during negative strand synthesis.

INHIBITION OF CELLULAR PROTEIN SECRETION BY POLIOVIRUS PROTEINS 2B AND 3A

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

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.

The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-γ Locus

Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theilers [cleanup Salmonella not Theilers]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theilers virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.

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.

Six RNA viruses and forty-one hosts: Viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

Poliovirus 3A Protein Limits Interleukin-6 (IL-6), IL-8, and Beta Interferon Secretion during Viral Infection

ABSTRACT During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.