Viral Hemorrhagic Septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the northern hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral genes expression. By systematically screening each of the six VHSV structural and nonstructural genes, we have identified matrix protein (M) as its most potent anti-host protein. VHSV-IVb M alone suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active SV40 promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters, and decreased RNAP II CTD Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I-III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M protein of a particular VHSV-Ia strain, F1, was significantly less potent than -IVb M at inhibiting SV40/luc expression, yet differed by just four amino acids. Mutation of D62 to alanine alone, or in combination with an E181 to alanine mutation (D62A/E181A), dramatically reduced the ability of -IVb M to suppress host transcription. Introducing either M D62A or D62A/E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced anti-transcriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. Importance: Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish Novirhabdovirus, VHSV, as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.
more »
« less
This content will become publicly available on May 1, 2024
Intracellular bottlenecking permits no more than three tomato yellow leaf curl virus genomes to initiate replication in a single cell
Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.
more » « less- Award ID(s):
- 1758912
- NSF-PAR ID:
- 10479650
- Editor(s):
- Wang, Aiming
- Publisher / Repository:
- PLoS Pathogens
- Date Published:
- Journal Name:
- PLOS Pathogens
- Volume:
- 19
- Issue:
- 5
- ISSN:
- 1553-7374
- Page Range / eLocation ID:
- e1011365
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Pagán, Israel (Ed.)more » « less
Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10−5substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.
-
Natural selection acts on cellular organisms by ensuring the genes responsible for an advantageous phenotype consistently reap the phenotypic advantage. This is possible because reproductive cells of these organisms are almost always haploid, separating the beneficial gene from its rival allele at every generation. How natural selection acts on plus-strand RNA viruses is unclear because these viruses frequently load host cells with numerous genome copies and replicate thousands of progeny genomes in each cell. Recent studies suggest that these viruses encode the Bottleneck, Isolate, Amplify, Select (BIAS) mechanism that blocks all but a few viral genome copies from replication, thus creating the environment in which the bottleneck-escaping viral genome copies are isolated from each other, allowing natural selection to reward beneficial mutations and purge lethal errors. This BIAS mechanism also blocks the genomes of highly homologous superinfecting viruses, thus explaining cellular-level superinfection exclusion.more » « less
-
more » « less
Summary The ability to edit plant genomes through gene targeting (
GT ) requires efficient methods to deliver both sequence‐specific nucleases (SSN s) and repair templates to plant cells. This is typically achieved usingAgrobacterium T‐DNA , biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum (tobacco) andSolanum lycopersicum (tomato), greater than 10‐fold enhancements inGT frequencies have been achieved usingDNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSN s and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV ). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR /Cas9 nucleases and repair templates achievedGT at an endogenousubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR ) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDV replicons, multiplexedGT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGT usingWDV ‐basedDNA replicons will make it possible to edit complex cereal genomes without the need to integrateGT reagents into the genome. -
ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.more » « less
