Defective Interfering Particles Research Articles

P.3.a.010 Risperidone long-acting injection: a one year observational study in a naturalistic setting

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement but persistent, innocuous infections

Research on cultivated shrimp suggests that they have the capability to tolerate viral pathogens in a highly specific manner by mechanisms currently unknown. The phenomenon is difficult to study in detail because they have a generation time of 1–2 yr and lack continuous cell lines. Thus, we developed a mosquito-densovirus model to examine whether similar phenomena occur in insects. Serial challenge of five generations with a stock densovirus ( AThDNV) resulted in progressive survival increases from 15% to 58%. Prevalence of AThDNV infection in surviving mosquito larvae (confirmed by PCR, histology, in situ hybridization and transmission electron microscopy) was relatively high (e.g. 36% in F2) but they grew normally to establish each succeeding generation. At the end of five generations, comparison of deduced amino acid sequences from genome fragments revealed a significantly higher ( p = 0.0 2 ) estimated prevalence of defective targets in the survivor virus population (29.7%±10.0 SD) than in the stored viral population (3.3%±5.8 SD). The results paralleled those reported for serially passaged C6/36 mosquito cell cultures infected with a densovirus. There, reduced infection rates are ascribed to the production of defective interfering particles (DIP). Thus, it is possible that the presence of prior AThDNV infections with a high level of DIP contributed to improved survival in our challenged F4 mosquito population. If so, it suggests that persistent viral infections in arthropods may serve in a specific, adaptive manner to reduce the incidence and severity of disease.

Structure and prevalence of replication silencer–3′ terminus RNA interactions in Tombusviridae

Tombusviridae is a large positive-strand RNA virus family. Tomato bushy stunt virus (TBSV), the type virus of this family, has a genome ending with AGCCC −OH, termed the 3′-complementary silencer sequence (3′CSS). The 3′CSS is able to base pair with a complementary internally-located sequence, 5′GGGCU, called the replication silencer element (RSE). In TBSV, previous compensatory mutational analysis of the RSE–3′CSS interaction showed it to be functionally important for viral RNA synthesis both in vitro and in vivo. However, these investigations also revealed that the RSE and 3′CSS are very sensitive to nucleotide changes, even when base pairing potential between the two elements is maintained. Consequently, an alternative investigative approach was used in this study where the wild-type sequences of these elements were preserved and their surrounding contexts were modified. Results from these analyses, using a TBSV DI RNA, revealed important new structural requirements necessary for the RSE and 3′CSS to operate in vivo. Collectively, the data suggest that accessibility of the elements and their proximity to adjoining stem structures are important functional parameters. Based on these findings, a working structural model for the TBSV RSE–3′CSS interaction is proposed that involves coaxial stacking of adjacent helices at either end of the RSE–3′CSS interaction. Components of this structural model are extendable to potential RSE–3′CSS interactions that were identified throughout Tombusviridae by comparative sequence analysis. This survey also revealed a significant level of diversity and modularity with respect to RSEs, 3′CSSs and their structural contexts and, moreover, suggests that RSE–3′CSS interactions are prevalent in Tombusviridae and related viruses.

Identification of sequence elements of tombusvirus-associated defective interfering RNAs required for symptom modulation

Defective interfering (DI) RNAs of tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5' proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.

Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

The mouse hepatitis virus (MHV) genome's 3' untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3'-terminal 42 nucleotides [3'(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3'(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3'-terminal 42-nucleotide element. Additionally we have identified a role for the 3'-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3'(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3' end of the MHV genome in subgenomic RNA synthesis.

Stem-Loop IV in the 5′ Untranslated Region Is a cis -Acting Element in Bovine Coronavirus Defective Interfering RNA Replication

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.

Torovirus non-discontinuous transcription: mutational analysis of a subgenomic mRNA promoter.

Toroviruses (order Nidovirales) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3'-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5' end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3' end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5'-ACN3-4CUUUAGA-3', within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21:6571-6580, 2002). We have now studied the--for nidoviruses unusual--non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5'-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

494. Assessing Lentiviral Vector Quality Using Transmission Electron Microscopy

Top of pageAbstract HIV-1 vectors are currently being pursued for a number of gene therapy applications. The infectious titer of HIV-1 vectors may be influenced by the presence of transduction inhibitors and defective interfering particles (DIPs) in vector preparations. To evaluate whether the mode of vector production or the nature of the pseudotyping envelope affects the levels of transduction inhibiting factors, we analyzed HIV-1 vectors pseudotyped with Vesicular Stomatitis Virus G-protein (VSVG) or Ross River Virus (RRV) envelope protein, prepared by ultracentrifugation or ultrafiltration, in the presence and absence of serum by Transmission Electron Microscopy (TEM). For TEM studies, the HIV-1 vector, CS-CGW, was mixed with latex beads of known size and concentration in equal proportions, centrifuged onto formvar coated grids and negative stained with Uranyl Acetate (UA) or Nanovan. HIV-1 particles were identified in TEM by size and morphology. Analysis of particle size distribution in vector preparations indicates that all vectors contain particles that are smaller in size (30-80 nm) in addition to the expected 80-120 nm retroviral particles. Size distribution analysis of the VSVG pseudotyped CSCGW vector (kindly provided by Philip Zoltick, Philadelphia, PA) prepared in the absence of serum shows that the unconcentrated vector contains 72% smaller size particles and 28% of the correct sized retroviral particles whereas vector samples concentrated by either ultracentrifugation or ultrafiltration show higher amounts (73%) of the correct sized particles (approximately 50-100 particles were analyzed in three independent grid spaces). The particle size distribution of the CSCGW vector prepared in the presence of serum is similar to that of the serum free vector (73% small particles/27% retroviral particles). Particle size distribution analysis of unconcentrated CSCGW vector pseudotyped with RRV shows a four fold lower amount of smaller particles than the VSVG-pseudotyped vector (19%/RRV vs. 72 %/VSVG). These results suggest that concentration by either centrifugation or filtration removes significant amount of small sized particles from vector preparations and increases the percentage of correct sized particles. Also the nature of the pseudotyping envelope may influence particle size. In conclusion, analysis of HIV-1 vectors by TEM suggests that production methods affect factors that influence vector transduction efficiency.

PROPERTIES OF A UNIQUE MUTANT OF HELICOVERPA ARMIGERA SINGLE-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS THAT EXHIBITS A PARTIAL MANY POLYHEDRA AND FEW POLYHEDRA PHENOTYPE ON EXTENDED SERIAL PASSAGING IN SUSPENSION CELL CULTURES

Serial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.

Defective Interfering RNA Hinders the Activity of a Tombusvirus-Encoded Posttranscriptional Gene Silencing Suppressor

Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

The growth of attenuated influenza vaccine donor strains in continuous cell lines

The growth of the Russian live attenuated influenza vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and B/USSR/60/69 was studied in cells of the VERO and Madin–Darby canine kidney (MDCK) lines as six-well cultures and cell factories infected at different multiplicities of infection. Yields for A/Leningrad/134/17/57 and A/Leningrad/134/47/57 were comparable in either cell line over a range of multiplicities but were about 10-fold lower than in the allantoic fluids of infected chicken embryos. For both A/Leningrad/134/47/57 and B/USSR/60/69, yields from the MDCK line were about 10-fold higher than for the VERO line. For B/USSR/60/69, yields in eggs were approximately 100-fold higher than those obtained in the MDCK line. A feature of the growth of B/USSR/60/69 was its reduced capacity to produce infectious progeny in either cell line at multiplicities of infection of 2.0 or 1.0 pfu/cell. Inhibition was due probably due to the presence of defective-interfering particles and was not detected with A/Leningrad/134/17/57 or A/Leningrad/134/47/57 in cultures of either line infected at the same multiplicities. Yields for both A/Leningrad/134/47/57 and B/USSR/60/69 in cells of the MDCK line were comparable when grown in six-well cultures or cell factories.

Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA.

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

Plant virus satellite and defective interfering RNAs: new paradigms for a new century.

Although many subviral RNAs reduce or intensify disease symptoms caused by the helper virus, only recently have clues concerning the mechanism of disease modulation been revealed. New models for DI RNA-mediated reduction in helper virus levels and symptom attenuation include DI RNA enhancement of posttranscriptional gene silencing (PTGS), which is an antiviral defense mechanism in plants. Symptom enhancement by the satRNA of Cucumber mosaic virus is caused by minus-strand induction of the programmed cell death pathway. In contrast, symptom enhancement by satC of Turnip crinkle virus is due to satC interference with virion formation, leading to increased levels of free coat protein, which is the viral suppressor of PTGS. Mutualism between satRNA and helper virus can be seen for the satRNA of Groundnut rosette virus, which contributes to the virus by allowing virion assembly. These novel findings are leading to re-evaluation of the relationships between subviral RNAs, helper viruses, and hosts.

The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA.

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an 'all-or-none' behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

Phosphorylation of vesicular stomatitis virus phosphoprotein P is indispensable for virus growth.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxy-terminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus recovery, kinetics of growth, and mRNA and protein synthesis were examined. We observed that virus recovery was completely abolished when all three phosphate acceptor sites in domain I or both sites in domain II were replaced with alanine. Single or double mutations in domain I (with the exception of P60/64) or single mutations in domain II had no adverse effect on virus recovery. VSVP227, carrying alanine at position 227, showed reduced kinetics of virus growth but increased kinetics of viral mRNA synthesis in infected cells. More interestingly, this particular virus exhibited a significantly reduced cytopathic effects and apoptosis in infected cells, implying that P may be involved in these processes. Furthermore, we found that DI RNAs of different sizes were generated by high-multiplicity passaging of various mutant VSVs, indicating that the viral RdRp may play a significant role in the process of DI particle generation. Taken together, our results suggest that the phosphorylation of residues in domains I and II of VSV P is indispensable for virus growth.

Interfering vaccine: a novel antiviral that converts a potentially virulent infection into one that is subclinical and immunizing

Infection with human influenza A virus can reach cataclysmic levels, with 40 or more million deaths arising from the 1918 pandemic. Preventative and therapeutic measures have improved since that time, but new approaches are needed. Here, we describe one such new approach—the interfering vaccine, which has two activities—it prophylactically prevents influenza, and at the same time converts an otherwise lethal infection into one that is avirulent and immunizing. Mice treated in this way develop a solid immunity that protects them against a subsequent challenge with homologous virus, and to a lesser extent from challenge with heterologous influenza A viruses. The interfering vaccine comprises non-infectious, defective interfering (DI) influenza A virus. Prophylaxis is mediated directly by DI RNA, and results from interference with the replication of the infecting virus. However, interference is incomplete, and there is sufficient wild-type virus multiplication to stimulate a virus-specific natural immunity. As the replication mechanism is common to all influenza A viruses, an interfering vaccine should protect from, and permit immunity to be developed to all influenza A viruses. Indeed, we demonstrate protection against two viruses with antigenically unrelated HA and NA proteins. Thus an interfering vaccine, unlike the conventional vaccine, is independent of the antigenicity of the infecting virus. In principle, interfering vaccines derived from other virus systems could also be developed.

The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: implications for the mechanism of RNA recombination.

RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase "landing" during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.

Analysis of the 3' cis-acting elements of rubella virus by using replicons expressing a puromycin resistance gene.

A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3' cis-acting elements (3' CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3' CSE varied. The 3' region necessary for optimal relative survival consisted of the 3' 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3' CSE. Within the 3' CSE, deletions in the approximately 245 nt that overlap the 3' end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the approximately 60-nt 3' untranslated region (UTR) were lethal. None of the 3' CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5' proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3' CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3' CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3' CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.

Effect of the Host and Temperature on the Formation of Defective RNAs Associated with Broad bean mottle virus Infection

ABSTRACT Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL.

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.