RNA recombination in brome mosaic virus: effects of strand-specific stem-loop inserts.

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.

Studies on functional interaction between brome mosaic virus replicase proteins during RNA recombination, using combined mutants in vivo and in vitro.

Two viral proteins, 1a and 2a, direct replication of brome mosaic bromovirus (BMV) RNAs as well as they participate in BMV RNA recombination. To study the relationship between replication and recombination, double BMV variants that carried mutations in 1a and 2a genes were tested. The observed effects revealed that the 1a helicase and 2a N-terminal or core domains were functionally linked during both processes in vivo. The use of a series of mutant BMV replicase (RdRp) preparations demonstrated in vitro the participation of the 1a and 2a domains in BMV RNA copying and in template switching during minus-strand synthesis. The observed effects support previous observations that the characteristics of homologous and nonhomologous recombination can be modified separately by mutations at different sites on BMV replicase proteins.

Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus.

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

Structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene.

We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. Based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.

Mutations in the helicase-like domain of protein 1a alter the sites of RNA-RNA recombination in brome mosaic virus.

A system that uses engineered heteroduplexes to efficiently direct in vivo crossovers between brome mosaic virus (BMV) RNA1 and RNA3 (P. Nagy and J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993) has been used to explore the possible involvement of BMV 1a protein, an essential RNA replication factor, in RNA recombination. Relative to wild-type 1a, several viable amino acid insertion mutations in the helicase-like domain of BMV 1a protein affected the nature and distribution of crossover sites in RNA3-RNA1 recombinants. At 24 degrees C, mutants PK19 and PK21 each increased the percentage of asymmetric crossovers, in which the RNA1 and RNA3 sites joined by recombination were not directly opposite each other on the engineered RNA3-RNA1 heteroduplex used to target recombination but rather were separated by 4 to 85 nucleotides. PK21 and another 1a mutant, PK14, also showed increases in the fraction of recombinants containing nontemplated U residues at the recombination junction. At 33 degrees C, the highest temperature that permitted infections with PK19, which is temperature sensitive for RNA replication, the mean location of RNA1-RNA3 crossovers in recombinants recovered from PK19 infections was shifted by nearly 25 bp into the energetically less stable side of the RNA1-RNA3 heteroduplex. Thus, mutations in the putative helicase domain of the 1a protein can influence BMV RNA recombination. The results are discussed in relation to models for recombination by template switching during pausing of RNA replication at a heteroduplexed region in the template.

Foreign complementary sequences facilitate genetic RNA recombination in brome mosaic virus.

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3' noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc. Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation codon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3' marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.

[Analysis of brain tumors in children from the Rzeszów region]. / Analiza guzów mózgu u dzieci z regionu Rzeszowskiego.

The authors present an analysis of brain tumors diagnosed in the pediatric ward of the voivodship hospital in Rzeszów between 1988 and 1992. Age, sex and clinical manifestation leading to hospitalisation, localisation and histopathology of the tumors were assessed.

Mutational analysis of the coat protein gene of brome mosaic virus: effects on replication and movement in barley and in Chenopodium hybridum.

The coat protein (CP) open reading frame (ORF) of brome mosaic virus (BMV) has been mutated to study host-related CP functions in barley, a systemic host, and in Chenopodium hybridum L. which supports both local lesion formation and systemic spread of BMV. To test the role of the N-terminal region of CP, mutants C1 to C3, which synthesized the CP lacking first seven amino acids, and mutant D1, which had Trp 22 and Thr 23 replaced with Phe-Gly-Ser, were generated. C1 to C3 inhibited virus systemic spread in C. hybridum but not in barley while D1 only reduced virus accumulation in noninoculated leaves of C. hybridum. More internal CP regions were tested by mutation of Lys 63 to Leu (mutant SP3) and Lys 129 to Arg (mutant SP1). SP1 behaved similarly to C1 to C3 while SP3 similarly to D1. In addition, SP3 reduced concentrations of RNA3 and RNA4 in both hosts. Apparently, various CP regions differentially affect, either directly or indirectly, virus translocation in different hosts, suggesting both the CP and host factors to be important for virus spread. Larger deletions in the CP ORF (mutants BB4 and SX1) or a decrease of CP production by using a frameshift mutant C, inhibited virus systemic spread in both hosts, and delayed the appearance of smaller local lesions on C. hybridum. Thus, the CP is not required for cell-to-cell movement but is required for systemic translocation of BMV.

[Diagnostic and therapeutic difficulties in a case of infection with Haemophilus influenzae]. / Trudnosci diagnostyczne i lecznicze w przypadku zakazenia Haemophilus influenzae.

A case is presented of generalized Haemophilus infection followed by its concentration in the central nervous system, joints and middle ear.

[A case of skin reactions to carbamazepine]. / Przypadek reakcji skórnych po karbamazepinie.

A case is described of severe cutaneous complications after carbamazepine administration. Attention is called to the frequency of adverse reactions to this drug.

The nucleotide sequence and genome organization of the RNA2 and RNA3 segments in broad bean mottle virus.

Complete nucleotide sequences of broad bean mottle virus (BBMV) genomic RNAs 2 and 3 were determined. They consist of 2811 and 2293 nucleotides, respectively. Both RNAs are caped and, unlike in other tricornaviruses, both initiate with an A residue. BBMV RNA2 is monocistronic and encodes an 815 amino acid 2a protein, whereas RNA3 is dicistronic, encoding for a 295 amino acid 3a protein and for the 190 amino acid coat protein. A central, 423 amino acid 2a protein core region is highly homologous among the three bromoviruses, whereas both N- and C-termini are more heterologous. Most of the homologies among 3a proteins are concentrated within the N-termini two-thirds of the molecule that is predominantly hydrophobic, whereas the C-terminal one-third contains a large number of charged amino acids. The homologies among coat proteins are clustered within several mostly hydrophobic, or neutral, domains. The 5' noncoding region of the RNA2 has 110 nucleotides, whereas that of RNA3 contains 330 nucleotides. As in cowpea chlorotic mottle virus, but unlike in Brome mosaic virus, the 5' noncoding region includes subgenomic promoter-like sequences. The BBMV RNA3 intercistronic region also has subgenomic promoter sequences and contains a long poly(A) stretch. At the 3' end, BBMV RNAs 2 and 3 have 257 and 236 noncoding nucleotides, respectively.

The nucleotide sequence and genome organization of the RNA-1 segment in two bromoviruses: broad bean mottle virus and cowpea chlorotic mottle virus.

The complete nucleotide sequences of the RNA-1 segments in broad bean mottle virus (BBMV) and cowpea chlorotic mottle virus (CCMV) were determined. BBMV RNA-1 consists of 3158 nucleotides and CCMV RNA-1 has 3171 nucleotides. Both BBMV and CCMV RNA-1 are capped at the 5' end but, unlike in other tricornaviruses, BBMV RNA-1 initiates with an A residue. Both BBMV and CCMV RNA-1 are monocistronic encoding for highly homologous 1a proteins of 966 and 958 amino acids, respectively. The highest homologies are clustered within two domains: the N-domain that aligns with the nsP1 Sindbis virus protein, a putative methyl transferase, and the C-domain which has a conserved nucleotide binding motif. Previous sequence comparisons suggest that the C-terminal domain may function as an NTP-dependent RNA helicase. In addition, we find that the C-domain has patterns similar to those of the reovirus and vaccinia virus guanylyl transferases. All this implies that 1a protein is a multifunctional polypeptide involved in both RNA capping and RNA polymerization processes.

Generation and analysis of nonhomologous RNA-RNA recombinants in brome mosaic virus: sequence complementarities at crossover sites.

All three single-stranded RNAs of the brome mosaic virus (BMV) genome contain a highly conserved, 193-base 3' noncoding region. To study the recombination between individual BMV RNA components, barley plants were infected with a mixture of in vitro-transcribed wild-type BMV RNAs 1 and 2 and an RNA3 mutant that carried a deletion near the 3' end. This generated a population of both homologous and nonhomologous 3' recombinant BMV RNA3 variants. Sequencing revealed that these recombinants were derived by either single or double crossovers with BMV RNA1 or RNA2. The primary sequences at recombinant junctions did not show any similarity. However, they could be aligned to form double-stranded heteroduplexes. This suggested that local hybridizations among BMV RNAs may support intermolecular exchanges.

Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs.

An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.