Plant Biotechnology-An Indispensable Tool for Crop Improvement.

Traditional plant breeding has helped to increase food production dramatically over the past five decades, and many countries have managed to produce enough food for the growing population, particularly in the developing world [...].

Studies on Improving the Efficiency of Somatic Embryogenesis in Grapevine (Vitis vinifera L.) and Optimising Ethyl Methanesulfonate Treatment for Mutation Induction.

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with ß-naphthoxyacetic acid (5 µM) and 6-benzylaminopurine (BAP-9.0 µM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar.

Increased Zygote-Derived Plantlet Formation through In Vitro Rescue of Immature Embryos of Highly Apomictic Opuntia ficus-indica (Cactaceae).

O. ficus-indica (prickly pear cactus) is an important forage and food source in arid and semiarid ecosystems and is the most important cactus species in cultivation globally. The high degree of apomixis in the species is a hindrance in plant breeding programs where genetic segregation is sought for the selection of superior genotypes. To understand if in ovulo embryo rescue could increase the proportion of zygotic seedlings, we compared the mature seed-derived seedlings with those regenerated from in vitro embryo rescue at 20, 25, 30, 35, and 40 post-anthesis days (PADs) in four Italian cultivars. The seedlings were classified as apomictic or zygotic based on molecular marker analysis using inter-sequence single repeat (ISSR) primers. Multiple embryos were recovered from all the cultured immature ovules, and plantlets were regenerated and acclimatized to the field post hardening, with success rates ranging from 62% ('Senza spine') to 83% ('Gialla'). The level of polyembryony differed among cultivars and recovery dates, with the highest being 'Rossa', producing 4.8 embryos/ovule at 35 PADs, and 'Gialla', the lowest, with 2.7 at 40 PADs. The maximum number of embryos observed within a single ovule was 14 in 'Trunzara bianca'. ISSR analysis revealed that ovule culture at 35 PADs produced the highest percentage of zygotic seedlings in all the cultivars, from 51% ('Rossa') to 98% ('Gialla'), with a high genotype effect as well. Mature seeds produced much fewer seedlings per seed, ranging from 1.2 in 'Trunzara bianca' to 2.0 in 'Rossa' and a lower percentage of zygotic seedlings (from 14% in 'Rossa' to 63% in 'Gialla'). Our research opens a pathway to increase the availability of zygotic seedlings in O. ficus-indica breeding programs through in ovulo embryo culture.

Management and Utilization of Plant Genetic Resources for a Sustainable Agriculture.

Despite the dramatic increase in food production thanks to the Green Revolution, hunger is increasing among human populations around the world, affecting one in nine people. The negative environmental and social consequences of industrial monocrop agriculture is becoming evident, particularly in the contexts of greenhouse gas emissions and the increased frequency and impact of zoonotic disease emergence, including the ongoing COVID-19 pandemic. Human activity has altered 70-75% of the ice-free Earth's surface, squeezing nature and wildlife into a corner. To prevent, halt, and reverse the degradation of ecosystems worldwide, the UN has launched a Decade of Ecosystem Restoration. In this context, this review describes the origin and diversity of cultivated species, the impact of modern agriculture and other human activities on plant genetic resources, and approaches to conserve and use them to increase food diversity and production with specific examples of the use of crop wild relatives for breeding climate-resilient cultivars that require less chemical and mechanical input. The need to better coordinate in situ conservation efforts with increased funding has been highlighted. We emphasise the need to strengthen the genebank infrastructure, enabling the use of modern biotechnological tools to help in genotyping and characterising accessions plus advanced ex situ conservation methods, identifying gaps in collections, developing core collections, and linking data with international databases. Crop and variety diversification and minimising tillage and other field practices through the development and introduction of herbaceous perennial crops is proposed as an alternative regenerative food system for higher carbon sequestration, sustaining economic benefits for growers, whilst also providing social and environmental benefits.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

Photosynthetic Parameters and Oxidative Stress during Acclimation of Crepe-Myrtle (Lagerstroemia speciosa (L.) Pers.) in a meta-Topolin-Based Micropropagation System and Genetic Fidelity of Regenerated Plants.

An improved and stable micropropagation system using the cytokinin, meta-Topolin (N6 (3-hydroxybenzylamino purine-mT), with nodal explants in Lagerstroemia speciosa L. was established. Among the different doses of mT, the maximum number of shoots with the highest shoot length was obtained using Murashige and Skoog's (MS) medium supplemented with 5.0 µM mT. The results were consistent throughout the proliferation period, when recorded at week 4, 8, and 12 of being cultured, with an average of 16.4 shoots per nodal explant, and having a mean length of 4.10 cm at week 8. Shoot proliferation rates could be further improved by a combination of 5.0 µM mT with 0.5 µM α-naphthalene acetic acid in MS medium; nodal explants produced an average of 24.3 shoots with a mean length of 5.74 cm after 8 weeks of being cultured. Among the five different concentrations of three auxins tested for the rooting of microshoots in MS medium, a 1.0 µM indole-3-butyric acid treatment was the best, with an average of 10.3 roots per microshoot at an average length of 3.56 cm in 93% of microshoots within 4 weeks of being transferred to this medium. A significant reduction of both chlorophyll a and b in leaves during the first week of acclimation corresponded with a high accumulation of malondialdehyde (MDH), indicating that lipid peroxidation affected chlorophyll pigments. From the second week of acclimation, photosynthetic pigment content significantly increased and MDH content decreased. The net photosynthetic rate and leaf carotenoid content showed almost linear increases throughout the acclimation period. Activity of antioxidant enzymes, namely, superoxide dismutase, catalase, and peroxidases, consistently increased throughout the acclimation period, corresponding with the accumulation of photosynthetic pigments, thus demonstrating the role of the improved antioxidant enzymatic defense system during acclimation. A comparison of parent plant DNA with that of the greenhouse acclimated plants using random amplified polymorphic DNA and inter-simple sequence repeat markers showed a monomorphic pattern indicating genetic stability and the suitability of the method for micropropagation of L. speciosa.

Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit 'Yuxiang' (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis 'Jinmi', respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.

Resistance of New Zealand Provenance Leptospermum scoparium, Kunzea robusta, Kunzea linearis, and Metrosideros excelsa to Austropuccinia psidii.

Resistance to the pandemic strain of Austropuccinia psidii was identified in New Zealand provenance Leptospermum scoparium, Kunzea robusta, and K. linearis plants. Only 1 Metrosideros excelsa-resistant plant was found (of the 570 tested) and no resistant plants of either Lophomyrtus bullata or L. obcordata were found. Three types of resistance were identified in Leptospermum scoparium. The first two, a putative immune response and a hypersensitive response, are leaf resistance mechanisms found in other myrtaceous species while on the lateral and main stems a putative immune stem resistance was also observed. Both leaf and stem infection were found on K. robusta and K. linearis plants as well as branch tip dieback that developed on almost 50% of the plants. L. scoparium, K. robusta, and K. linearis are the first myrtaceous species where consistent infection of stems has been observed in artificial inoculation trials. This new finding and the first observation of significant branch tip dieback of plants of the two Kunzea spp. resulted in the development of two new myrtle rust disease severity assessment scales. Significant seed family and provenance effects were found in L. scoparium, K. robusta, and K. linearis: some families produced significantly more plants with leaf, stem, and (in Kunzea spp.) branch tip dieback resistance, and provenances provided different percentages of resistant families and plants. The distribution of the disease symptoms on plants from the same seed family, and between plants from different seed families, suggested that the leaf, stem, and branch tip dieback resistances were the result of independent disease resistance mechanisms.

Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips.

Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated species. The number of vegetatively propagated species conserved by cryopreservation is increasing through development of vitrification-based methods; droplet vitrification in particular is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. This research investigated if cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thence aid in developing a droplet vitrification protocol for kiwifruit using Actinidia chinensis var. chinensis 'Hort16A' as a model. Cold acclimation of donor plantlets at 4 °C for 2 weeks followed by sucrose pre-culture of shoot tips and supplementation of ascorbic acid (0.4 mM) in all media throughout the procedure registered 40% regeneration after cryopreservation. Transmission electron microscope imaging of meristematic cells confirmed sucrose and ascorbic acid pre-treatment of shoot tips from cold acclimated plantlets following treatment in vitrification solution exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast cells without cold acclimation or sucrose and ascorbic acid pre-treatments exhibited minimal change after exposure to vitrification solution. After cryopreservation and recovery, all cells of untreated shoot tips showed rupture of the plasma membrane, loss of cytoplasmic contents and organelle distortions. By comparison, most pre-treated shoot-tip cells from cold acclimated plantlets retained their structural integrity, showing that only those cells that have been dehydrated and plasmolysed can withstand cryopreservation by vitrification.

Cell death patterns in Arabidopsis cells subjected to four physiological stressors indicate multiple signalling pathways and cell cycle phase specificity.

Corpse morphology, nuclear DNA fragmentation, expression of senescence-associated genes (SAG) and cysteine protease profiles were investigated to understand cell death patterns in a cell cycle-synchronised Arabidopsis thaliana cell suspension culture treated with four physiological stressors in the late G2 phase. Within 4 h of treatment, polyethylene glycol (PEG, 20 %), mannose (100 mM) and hydrogen peroxide (2 mM) caused DNA fragmentation coinciding with cell permeability to Evans Blue (EB) and produced corpse morphology corresponding to apoptosis-like programmed cell death (AL-PCD) with cytoplasmic retraction from the cell wall. Ethylene (8 mL per 250-mL flask) caused permeability of cells to EB without concomitant nuclear DNA fragmentation and cytoplasmic retraction, suggesting necrotic cell death. Mannose inducing glycolysis block and PEG causing dehydration resulted in relatively similar patterns of upregulation of SAG suggesting similar cell death signalling pathways for these two stress factors, whereas hydrogen peroxide caused unique patterns indicating an alternate pathway for cell death induced by oxidative stress. Ethylene did not cause appreciable changes in SAG expression, confirming necrotic cell death. Expression of AtDAD, BoMT1 and AtSAG2 genes, previously shown to be associated with plant senescence, also changed rapidly during AL-PCD in cultured cells. The profiles of nine distinct cysteine protease-active bands ranging in size from ca. 21.5 to 38.5 kDa found in the control cultures were also altered after treatment with the four stressors, with mannose and PEG again producing similar patterns. Results also suggest that cysteine proteases may have a role in necrotic cell death.

Novel antiviral activity of l-dideoxy bicyclic nucleoside analogues versus vaccinia and measles viruses in vitro.

Dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with d-chirality have previously been described by us to inhibit replication of human cytomegalovirus. We herein report for the first time that activity against vaccinia virus (VACV) was achieved using novel l-analogues. A structure-activity relationship was established: Antiviral activity versus VACV was highest with an ether side chain with an optimum of n-C(9)H(18)-O-n-C(5)H(11). This gave an IC(50) of 190 nM, a 60-fold enhancement over the FDA-approved antiviral cidofovir. Interestingly, l-ddBCNAs also inhibit wild type measles virus syncytia formation with a TCID(50) of 7.5 µM for the lead compound. We propose that l-ddBCNAs represent significant innovative antiviral candidates versus measles and poxviruses, and we suggest a mechanism of action versus one or more cellular targets that are essential for viral replication.

Biofortification of tomato (Solanum lycopersicum) fruit with the anticancer compound methylselenocysteine using a selenocysteine methyltransferase from a selenium hyperaccumulator.

Methylselenocysteine (MeSeCys) is an amino acid derivative that possesses potent anticancer activity in animals. Plants that can tolerate growth on soils with high Se content, known as Se hyperaccumulators, do so by converting inorganic Se to MeSeCys by the enzyme selenocysteine methyltransferase (SMT). A cDNA encoding the SMT from a Se hyperaccumulator was overexpressed in tomato (Solanum lycopersicum). Transgenic plants were provided with selenite or selenate to the roots during fruit development, and liquid chromatography-mass spectrometry was used to show that MeSeCys accumulated in the fruit but not in the leaves. Depending on the transgenic line and Se treatment, up to 16% of the total Se in the fruit was present as MeSeCys. MeSeCys was produced more effectively from selenite on a percentage conversion basis, but greater accumulation of MeSeCys could be achieved from selenate due to its better translocation from the roots. MeSeCys was heat stable and survived processing of the fruit to tomato juice.

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora.

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0-32.6% of cultured ovary pieces and 0-18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.

Accumulation of an organic anticancer selenium compound in a transgenic Solanaceous species shows wider applicability of the selenocysteine methyltransferase transgene from selenium hyperaccumulators.

Tolerance to high selenium (Se) soils in Se-hyperaccumulating plant species is correlated with the ability to biosynthesise methylselenocysteine (MeSeCys), due to the activity of selenocysteine methyltransferase (SMT). In mammals, inclusion of MeSeCys in the diet reduces the incidence of certain cancers, so increasing the range of crop plants that can produce this compound is an attractive biotechnology target. However, in the non-Se accumulator Arabidopsis, overexpression of SMT does not result in biosynthesis of MeSeCys from selenate because the rate at which selenate is reduced to selenite by ATP sulfurylase (ATPS) is low. This limitation is less problematic in other species of the Brassicaceae that can produce MeSeCys naturally. We investigated the potential for biosynthesis of MeSeCys in other plant families using Nicotiana tabacum L., a member of the Solanaceae. When plants were watered with 200 microM selenate, overexpression of a SMT transgene caused a 2- to 4-fold increase in Se accumulation (resulting in increased numbers of leaf lesions and areas of necrosis), production of MeSeCys (up to 20% of total Se) and generation of volatile dimethyl diselenide derived directly from MeSeCys. Despite the greatly increased accumulation of total Se, this did not result in increased Se toxicity effects on growth. Overexpression of ATPS did not increase Se accumulation from selenate. Accordingly, lines overexpressing both ATPS and SMT did not show a further increase in total Se accumulation or in leaf toxicity symptoms relative to overexpression of SMT alone, but directed a greater proportion of Se into MeSeCys. This work demonstrates that the production of the cancer-preventing compound MeSeCys in plants outside the Brassicaceae is possible. We conclude that while the SMT gene from Se hyperaccumulators can probably be utilised universally to increase the metabolism of Se into MeSeCys, the effects of enhancing ATPS activity will vary depending on the species involved.

Preclinical development of bicyclic nucleoside analogues as potent and selective inhibitors of varicella zoster virus.

OBJECTIVES: To progress the anti-varicella-zoster-virus (VZV) aryl bicyclic nucleoside analogues (BCNAs) to the point of Phase 1 clinical trial for herpes zoster. METHODS: A new chromatography-free synthetic access to the lead anti-VZV aryl BCNAs is reported. The anti-VZV activity of lead Cf1743 was evaluated in monolayer cell cultures and organotypic epithelial raft cultures of primary human keratinocytes. Oral dosing in rodents and preliminary pharmacokinetics assessment was made, followed by an exploration of alternative formulations and the preparation of pro-drugs. We also studied uptake into cells of both parent drug and pro-drug using fluorescent microscopy and biological assays. RESULTS: Cf1743 proved to be significantly more potent than all reference anti-VZV compounds as measured either by inhibition of infectious virus particles and/or by viral DNA load. However, the very low water solubility of this compound gave poor oral bioavailability (approximately 14%). A Captisol admixture and the 5'-monophosphate pro-drug of Cf1743 greatly boosted water solubility but did not significantly improve oral bioavailability. The most promising pro-drug to emerge was the HCl salt of the 5'-valyl ester, designated as FV-100. Its uptake into cells studied using fluorescent microscopy and biological assays indicated that the compound is taken up by the cells after a short period of incubation and limited exposure to drug in vivo may have beneficial effects. CONCLUSIONS: On the basis of its favourable antiviral and pharmacokinetic properties, FV-100 is now being pursued as the clinical BCNA candidate for the treatment of VZV shingles.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: influence on protein stability and activity.

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.

Discovery of a new family of inhibitors of human cytomegalovirus (HCMV) based upon lipophilic alkyl furano pyrimidine dideoxy nucleosides: action via a novel non-nucleosidic mechanism.

Following our discovery of the potent anti-varicella zoster virus action of lipophilic alkyl furano pyrimidine 2'-deoxynucleosides, we now report that 2',3'-dideoxy sugar analogues are devoid of anti-VZV activity but are potent and selective inhibitors of human cytomegalovirus (HCMV). The present compounds are active in vitro at ca. 1 microM with cytotoxicity only above 200 microM. Importantly, we have discovered that the new agents do not act as nucleoside analogues, despite their nucleosidic structure, and time of addition studies revealed that the compounds may inhibit HCMV at an event in the replication cycle of the virus that precedes DNA synthesis. They represent new leads in the discovery of improved therapies for HCMV, particularly in view of their novel mechanism of action.