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Viral invasion and host defense: strategies and counter-strategies James C Carrington∗ and Steven A Whitham

The outcome of infection of plants by viruses is determined pendent RNA polymerase activity and other associated by the net effects of compatibility functions and defense activities (e.g. helicase, capping, priming) in close asso- responses. Recent advances reveal that viruses have the ciation with cytoplasmic surfaces of membranes [2]. The capacity to modulate host compatibility and defense functions docking of replication complexes to membranes appears by a variety of mechanisms. to be a point of intimate interaction between virus and host cell. In several cases, complexes associate specifically with endoplasmic reticulum (ER)-derived membranes Addresses or vesicles through specific membrane-binding functions Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA of replication proteins [3,4]. The basis for membrane ∗e-mail: [email protected] specificity, however, remains obscure. The participation of cellular factors, such as host proteins or protein complexes, Current Opinion in Plant Biology 1998, 1:336–341 has been a point of speculation for many years, with http://biomednet.com/elecref/1369526600100336 only limited direct evidence to support functional roles  Current Biology Ltd ISSN 1369-5266 of host factors [2]. This may change in the near future through the clever use of yeast as an adopted host for Abbreviations brome mosaic virus (BMV) to analyze RNA replication; BMV brome mosaic virus CaMV cauliflower mosaic virus Paul Ahlquist and colleagues have developed selectable ER endoplasmic reticulum and counter-selectable BMV RNA-based replicons for HC-Pro helper component-proteinase yeast [5•,6,7]. For example, BMV replicons expressing HR hypersensitive response URA3 allow ura3 yeast to grow in the absence of uracil mab maintenance of BMV functions MP movement protein and thus provide a positive selection for cells with PSBMV pea seedborne mosaic potyvirus active BMV replication. Conversely, URA3 confers a lethal PVX potato virus X phenotype in the presence of 5-fluoroortic acid, and thus TBRV tomato black ring nepovirus provides a selection for yeast cells that lose the ability TEV tobacco etch virus TMV tobacco mosaic virus to support BMV replication. The characteristics of BMV VIGS viral induced gene silencing replicon amplification are remarkably similar in yeast cells and plant cells, providing a rationale for applying yeast genetics to identify host factors that influence Introduction replication. The MAB1, MAB2 and MAB3 (Maintenance of When discussing susceptibility or resistance of plants to BMV functions) loci contribute distinct functions to BMV viruses, it is convenient to consider ‘susceptibility factors’ genomic RNA replication, subgenomic RNA synthesis and ‘resistance factors’ as separate entities governing and accumulation of BMV-encoded replication proteins •• infection. It can be argued, however, that the distinction [8 ]. Recessive mutations at each locus condition a between susceptibility and resistance to viruses may not temperature-sensitive yeast growth phenotype, suggesting be so straightforward. We are particularly intrigued by that the wild-type gene products are necessary for the the possibility that viruses can be active promoters of normal cellular function. Dissection of the roles of the susceptibility or suppressors of defense. In this review, MAB gene products in viral RNA replication will represent we will consider selected aspects of susceptibility, such a significant milestone in understanding compatibility as virus–host interactions that govern virus genome during virus–host interactions. replication and intercellular movement, and a limited discussion of defense responses. We will also summarize DNA-containing viruses, such as the geminiviruses, repli- recent findings, as well as our views, on how viruses cate in the host plant nucleus where the cellular DNA may modulate host functions to ensure their own efficient replication apparatus is employed. The geminiviruses replication and movement. use host DNA polymerase and associated factors in conjunction with a virus-encoded replication protein Virus–host interactions: susceptibility factors (termed RepA or AL1). The RepA/AL1 protein provides A plant host is considered fully susceptible to a given virus several key activities that integrate viral and host functions if the virus can successfully complete three processes: controlling early gene expression, late gene expression and genome replication, cell-to-cell movement (local) and DNA replication [9,10]. This protein binds to the stem long-distance (vasculature-dependent) movement [1]. For structure at the replication origin and nicks the DNA prior most RNA-genome viruses, RNA replication is catalyzed to initiation of rolling-circle DNA replication [9,11–13]. by a core set of virus-encoded enzymes with RNA-de- Like many animal-infecting DNA viruses that use the Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham 337

cellular DNA replication apparatus, the geminiviruses face therefore, must overcome the lack of DNA replication a serious problem; they infect terminally differentiated, factors in G0 cells. Two significant breakthroughs have resting state (G0) cells, but these cells lack factors required shed light on possible mechanisms whereby geminiviruses for DNA replication ([14], and references therein). As solve this problem. First, geminiviruses induce expression detailed below, geminiviruses likely solve this problem of at least some of the components necessary for DNA through modulating factors that normally control the cell synthesis upon infection. Proliferating cell nuclear antigen cycle. (PCNA), a DNA polymerase accessory factor normally found only in S-phase cells, is induced in quiescent Virus-encoded susceptibility factors controlling cell-to- mesophyll cells by infection with tomato golden mosaic cell and long-distance movement are called ‘movement geminivirus (TGMV) [14]. Using AL1 transgenic plants, proteins’ (MPs) and have been reviewed extensively induction of PCNA was shown to require only the TGMV in recent years [1,15,16]. Movement proteins facilitate AL1 gene. Second, the AL1/RepA protein physically movement through interactions with the viral genome or interacts with host-encoded retinoblastoma-like tumor assembled virus particle, intracellular transport pathways suppressor proteins (pRbs) [28••,29–31]. Wheat dwarf and structures, and plasmodesmata. For geminiviruses, geminivirus mutants with substitutions affecting the prob- two MPs are required for cell-to-cell movement. One able pRb-like protein binding site of Rep exhibit defects (BR1) is necessary to transport viral ssDNA genomes in viral DNA synthesis [29], supporting the concept between the nucleus and the cytoplasm [17,18]. The that this interaction is necessary. The well-characterized BR1 protein, therefore, is a nuclear shuttle protein with mammalian pRB protein serves as key G1 checkpoint nuclear import and export signals. The other (BL1) regulator, preventing completion of G1 and entry into interacts with ER-derived membranes and plasmodesmata S-phase [32]. Phosphorylation by cyclin-dependent ki- to facilitate transit of viral DNA to adjacent plant cells, nases relieves the cell cycle-inhibitory activity of pRb and possibly by modifying the plasmodesmata structure and allows progression into S-phase [32]. Plants, like mammals, transport properties [19,20]. Tobacco mosaic virus (TMV) likely use one or more pRb-like proteins as checkpoint MP interacts with viral RNA and modifies plasmodesmata inhibitors of the cell cycle. It is reasonable to suggest, [21–23]. The TMV MP was shown to interact with therefore, that geminivirus Rep protein interacts with, microtubules in infected or transfected cells [24,25], and it and inactivates (or at least diverts), pRb-like protein in was proposed that this might enable intracellular transport infected cells. This would relieve the transcriptional block of movement complexes from sites of genome replication to production of S-phase-specific mRNAs and provide to plasmodesmata. Recent data, however, suggest that a pool of enzymes and factors necessary for viral DNA the MP–microtubule association may occur relatively late replication. Thus, plant geminiviruses join the ranks in the infection process, possibly as part of a pathway of animal DNA tumor-inducing viruses, such as SV40 to dissipate high local concentrations or to degrade MP and adenovirus, which encode proteins that affect the [26]. Early during the infection process, TMV MP, like cell cycling apparatus through interactions with tumor the geminivirus BL1 [19], associates with ER-derived suppressor proteins [33]. membranes and plasmodesmata [26]. Might the association between the ER and diverse MPs from different viruses A novel defense response against viruses be telling us something important? Recall that plasmodes- Defense responses involving dominant resistance (R) mata contain an extension of the ER, the desmotubule, genes, hypersensitive cell death and induction of systemic which is threaded through the central channel [16,27]. acquired resistance have been reviewed nicely by others Quite possibly, transport complex movement through [34•,35•,36,37] and will not be covered here. Rather, we plasmodesmata requires specific interactions first with will focus on a novel defense response — post-transcrip- cortical ER and then with the desmotubule component in tional gene silencing — that has only recently been linked the channel. to natural forms of virus resistance.

Induction of host compatibility factors by Post-transcriptional gene silencing is a remarkable pro- viruses cess that has been reviewed extensively [38–41]. The Do viruses have the ability to promote their own infections hallmarks of post-transcriptional silencing of nuclear-de- by stimulation of host susceptibility factors or structures rived sequences include, among other features, high needed for genome replication, genome expression or transcription levels of the silenced gene but relatively low movement? Given that the number of cellular factors steady-state cytoplasmic levels of the transcript. Silencing shown to play definitive roles in promoting virus infection occurs through a homology-dependent mechanism and is small, there are relatively few examples to ponder. The functions in trans to suppress identical or closely related geminiviruses, however, provide an excellent case to argue sequences. Evidence suggests that silencing is due that viruses have the potential to modulate susceptibility. to activation of a sequence-specific RNA degradation apparatus, perhaps by a mechanism involving synthesis of As mentioned above, geminiviruses are dependent on complementary nucleic acids. Silencing of a nuclear-de- the host DNA replication apparatus for replication, and, rived sequence can be triggered by either nuclear genes 338 Plant–microbe interactions

or by cytoplasmic, replicating nucleic acids containing transient depletion of many, but not all, classes of host homologous sequences. Furthermore, a silenced state in cell mRNAs in pea embryos [50,51]. The host mRNA a plant can be generated systemically through transport of modulation occurs specifically in cells supporting virus a mobile signal [42••,43••]. replication, and exhibits some of the characteristics of the heat shock response, such as induction of HSP70 and Whereas it has been known for some time that viral polyubiquitin mRNA expression. Although the bases for sequences can induce silencing of an endogenous nuclear suppression and subsequent recovery of transcript levels gene or a transgene [44,45], it was only recently demon- are not clear, the consequences of inhibition of host cell strated that viruses can trigger silencing in the apparent mRNA accumulation on induction of defense responses absence of homologous nuclear sequences. Infection are potentially far-reaching. As most types of known of Nicotiana clevelandii by tomato blackring nepovirus induced defense responses require host gene expression (TBRV) strain W22 results in an initial symptomatic phase [34•], inhibition of mRNA accumulation or translation may in which the virus moves systemically, followed by a recov- severely limit the extent or timing of a defense program. ery state in which new tissue developing post-inoculation is asymptomatic and largely devoid of virus [46••]. Further, Additional lines of evidence suggest that potyviruses can silencing of the viral sequence is active in the recovered modulate host defense responses, and that modulation tissue, conferring effective resistance against W22 and a can condition enhanced accumulation and movement of closely related strain. Prolonged infection of Brassica napus potyviruses as well as heterologous viruses. Typically, by cauliflower mosaic virus (CaMV), a pararetrovirus, also in single-infections by a non-potyvirus, virus genome induces post-transcriptional gene silencing independent of amplification at the single-cell level shuts off after an homologous transgenes [47••]. These findings are highly exponential amplification phase within 12–24 hours of significant as they indicate that homology-dependent gene inoculation [52]. Tobacco etch virus (TEV), in contrast, silencing may be a general antiviral response. The logic accumulates at a slower initial rate but for considerably for such a response is impeccable: extreme resistance can longer periods of time (e.g., 72 hours post-inoculation; be elicited after infection by a broad range of infecting [53]). TEV mutants with defects in helper component- viruses using a general, adaptive silencing apparatus. An proteinase (HC-Pro) exhibit a premature amplification important unresolved issue, however, is the extent to shut-off phenotype by 24 hours post-inoculation and which viruses, other than those mentioned, elicit silencing are restricted in long-distance movement [54•]. These upon infection. Most compatible virus–host combinations observations suggest that wild-type HC-Pro may function do not show the recovery phenotypes as do TBRV and to suppress one or more induced defense responses. CaMV. It is possible, however, that silencing is triggered Significantly, when heterologous viruses are co-inoculated transiently to limit the extent of virus replication in with TEV or other potyviruses, or when heterologous infected tissue, thus lessening the extent of disease, viruses infect plants or cells in the presence of functional but not in time to limit spread systemically. Silencing HC-Pro, the heterologous viruses exhibit an enhanced as a general antivirus response in both compatible and genome accumulation phenotype [55,56,57•]. In the case incompatible virus–host combinations deserves careful of infections of potato virus X (PVX) expressing HC-Pro attention. or an HC-Pro polyprotein precursor, the enhancement of PVX is actually the result of continued amplification Do viruses fight back? beyond the point at which amplification normally shuts off If one examines the way animal viruses deal with innate [57•]. A plausible explanation for all of these results is that and adaptive immune system defenses, it becomes clear potyviruses, through a key activity of HC-Pro, suppress that these agents are masters of the counter-defensive induction of host defense responses that would normally strategy. For example, poxviruses encode several types result in early amplification shut-off and restricted ac- of cytokine receptor mimic proteins [48]. These atten- cumulation (Figure 1). The antiviral defense responses uate the inflammatory and immune responses normally could have broad-spectrum activity against a range of triggered by virus-infected cells. Also, numerous animal diverse viruses, or could be induced to have specificity viruses encode inhibitors of apoptosis proteins (IAPs), against many different viruses. The mechanistic details preventing normal immune signaling that occurs through of this system are not yet resolved, although recently activation of cell death pathways [49]. obtained evidence is consistent with HC-Pro mediating a suppression of homology-dependent gene silencing (KD Do plant viruses deploy counter-defensive strategies? Kasschau and JC Carrington, unpublished data). Recent findings offer suggestions that certain plant viruses are able to interdict defense mechanisms or alter host Finally, the potential influence of viruses on signal cell metabolism for their own benefit. In a series of transduction pathways controlling a wide range of cellular elegant experiments examining host cell transcript and activities is just now being realized. Some of the best protein levels in situ in tissue across an advancing ongoing work in this area deals with the virulence-attenu- infection front, Andy Maule and colleagues demonstrated ating hypovirus of the chestnut blight fungus, Cryphonectria that pea seedborne mosaic potyvirus (PSBMV) induces parasitica. Donald Nuss and co-workers have shown that Viral invasion and host defense: strategies and counter-strategies Carrington and Whitham 339

Figure 1

ER HC-Pro

Accumulation of Local VIGS or Systemic acquired viral RNAs other induced silencing or other defense responses systemic responses?

Viral RNA Amplification shut-off replication complex or viral RNA degradation Current Opinion in Plant Biology

Modulation of antiviral defense responses by potyvirus TEV HC-Pro. The effects of HC-Pro on accumulation and movement of TEV and heterologous viruses [54•,55,56,57•] suggest that HC-Pro protein may suppress local and systemic defense responses. The defense responses affected by HC-Pro may include virus-induced gene silencing (VIGS), induced (non-HR) reactions, systemic acquired silencing or other systemic responses. The dotted line indicates that there is currently no direct experimental evidence suggesting that HC-Pro does interfere with a systemic host defense response. The viral replication complex is comprised of virus-encoded and possibly host-encoded proteins (represented by gray balls), which synthesize viral RNAs in association with endoplasmic reticulum (ER) derived membranes. hypovirus infection suppresses a G-protein signaling cas- (HR- and non-HR-dependent) virus–host combinations cade through down-regulation of the G-protein a-subunit, will reveal loci necessary for recognition and response CPG-1 [58–60]. This G-protein signaling pathway nega- signaling during defense. Clearly, the successes in recovery tively regulates cAMP levels. Significantly, virus infection, of highly informative mutants with defects in the sig- transgenic co-suppression of CPG-1, and drug-induced naling pathways for defense against bacterial and fungal elevation of cAMP levels each result in effects consistently pathogens sets an encouraging precedence [61]. Second, associated with hypovirulence of the fungus, including loss-of-susceptibility mutants using normally compatible reduced asexual sporulation, reduced pigmentation and virus–host combinations will reveal loci necessary for altered colony morphology. The ramifications of induction genome replication and movement. Besides the yeast mab of a hypovirulent state by hypoviruses are not immediately mutants with defects in supporting BMV RNA replication, clear, although it is possible that an ecological advantage an Arabidopsis mutant (tom1) has been recovered with is afforded. As sexual reproduction of hypovirus-containing a defect in supporting TMV replication [62]. Recovery C. parasitica strains is suppressed, genetic recombination in of large numbers of susceptibility mutants, however, a hypovirulent population should be limited. This would may require particular finesse, as the cellular factors promote proliferation of uniform anastomosis compatibility and structure needed to support virus infection almost groups, which in turn would promote distribution of the certainly are critical for normal cellular function. We virus by its normal transmission route — hyphal fusion. believe that the necessary mutants are attainable, however, While the influence of hypovirus infection on signaling in through development of novel virus–host genetic systems C. parasitica is now becoming clear, the effects of plant that are amenable to high-throughput selections and viruses on G-protein or other signaling cascades has yet to screens using mass inoculation procedures and selectable be established. The tools are available, however, to address or screenable viruses. this issue in detail. Acknowledgements Conclusions and future prospects Work in the author’s laboratory was supported by grants from the National Institute of Health (GM18529 to SA Whitham; AI27832 to JC Carrington) Although we have witnessed some significant develop- and the United States Department of Agriculture (95-37303-1867). ments recently, we suggest that the most exciting days in the quest to understand compatibility and incompatibility References and recommended reading functions governing virus infection are still ahead of us. Papers of particular interest, published within the annual period of review, A major limitation at this point is the paucity of useful have been highlighted as: host mutants that exhibit altered susceptibility phenotypes • of special interest and that can serve as starting points for isolation of •• of outstanding interest relevant genes. Two types of host mutants should be pursued aggressively. First, gain-of-susceptibility, or 1. Carrington JC, Kasschau KD, Mahajan SK, Schaad MC: Cell-to- cell and long-distance movement of viruses in plants. Plant loss-of-resistance, mutants using normally incompatible Cell 1996, 8:1669-1681. 340 Plant–microbe interactions

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