MALT1 Inhibition of Oral Carcinoma Cell Invasion and ERK/MAPK Activation.

The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression.

Charge disproportionation and the pressure-induced insulator-metal transition in cubic perovskite PbCrO3.

The perovskite PbCrO3 is an antiferromagnetic insulator. However, the fundamental interactions leading to the insulating state in this single-valent perovskite are unclear. Moreover, the origin of the unprecedented volume drop observed at a modest pressure of P = 1.6 GPa remains an outstanding problem. We report a variety of in situ pressure measurements including electron transport properties, X-ray absorption spectrum, and crystal structure study by X-ray and neutron diffraction. These studies reveal key information leading to the elucidation of the physics behind the insulating state and the pressure-induced transition. We argue that a charge disproportionation 3Cr(4+) → 2Cr(3+) + Cr(6+) in association with the 6s-p hybridization on the Pb(2+) is responsible for the insulating ground state of PbCrO3 at ambient pressure and the charge disproportionation phase is suppressed under pressure to give rise to a metallic phase at high pressure. The model is well supported by density function theory plus the correlation energy U (DFT+U) calculations.

Stable deletions arising in the readthrough region of Soil-borne wheat mosaic virus RNA2 define the 5' limit of the functional promoter for the p19 subgenomic RNA.

The appearance of de novo deletion mutations in the readthrough (RT) region (nucleotide positions 861-2591) downstream of the capsid protein (CP) gene of a Japanese strain of Soil-borne wheat mosaic virus RNA2 was examined using infectious transcripts. Mutant RNA2s with different deletions predominated in independent serial passage experiments but all best-adapted mutants retained the 3'-terminal portion of the RT gene in frame with the CP gene. The longest best-adapted mutation deleted the 1434 nucleotides between positions 1061 and 2494. When the RT protein was truncated by insertion of a termination codon plus an additional nucleotide to give a +1 frame-shift, after serial passages the progeny viruses regained the ability to express the C-terminal region of RT by an internal deletion. The 5' terminus of the p19 subgenomic RNA was identified at position 2598 and an essential transcription signal for this mRNA mapped between positions 2534 and 2563. A mutant in which this essential promoter element has been deleted cannot transcribe the p19 subgenomic RNA and has lost infectivity in planta. These results indicate that the 3'-terminal region of the RT gene has a major function in cis for expression of p19, which is essential for infecting plants. A reason for retaining the RT C-terminal region in stable deletion mutants is still unknown.

Detection of rice grassy stunt tenuivirus nonstructural proteins p2, p5 and p6 from infected rice plants and from viruliferous brown planthoppers.

The genome of Rice grassy stunt virus (RGSV) consists of 6 ambisense RNA segments, among which RNAs 1, 2, 5 and 6 are equivalent to RNAs 1, 2, 3 and 4, respectively, of Rice stripe virus, the type species of the genus Tenuivirus. The RGSV 36-kDa nucleocapsid protein (N) is encoded on the complementary strand of RNA 5. Here, we studied accumulation of three nonstructural proteins, a 23-kDa p2 protein encoded on vRNA 2 (virus genomic strand), a 22-kDa p5 protein encoded on vRNA 5, and a 21-kDa p6 protein encoded on vRNA 6, from RGSV-infected rice leaf tissues and from viruliferous vector insects (brown planthopper, Nilaparvata lugens) by Western blot analyses. p2, p5 and p6 were detected from RGSV-infected rice leaf homogenates; p2 was detected mostly in the cytoplasmic soluble fraction but also a small amount was detected in the cell-wall, organelle-enriched and crude membrane fractions; p5 and p6 were detected from the cytoplasmic soluble fraction in large amounts. Among individual nymphs of N. lugens raised on RGSV-infected rice leaves, only 20% of insects were positive with the N protein. A large amount of p5 was detected from all the N-positive insects. Small amounts of p2 and p6 were detected only from a subset of the N- and p5-positive insects. p5 may have an essential role in virus infection in both plant and insect hosts, whereas p2 may function in plants such as a cell-to-cell movement protein.

Reassortment between genetically distinct Japanese and US strains of Soil-borne wheat mosaic virus: RNA1 from a Japanese strain and RNA2 from a US strain make a pseudorecombinant virus.

Soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, has a plus-sense bipartite RNA genome. Japanese and US strains of SBWMV are genetically distantly related, despite their biologically identical properties. Here we report formation of a pseudorecombinant virus consisting of RNA1 from a Japanese strain and RNA2 from a US strain, using infectious in vitro transcripts for both strains. Full-length infectious cDNA clones for a Japanese strain were previously constructed (Yamamiya and Shirako [38]). For RNA1 of a US strain, due to instability of full-length cDNA clones in Escherichia coli cells, it was necessary to prepare a full-length template DNA for in vitro transcription by combining overlapping 5'-terminal and 3'-terminal cDNAs individually cloned in two plasmids, whereas for RNA2 a full-length cDNA clone was the template. For infectivity assays, Chenopodium quinoa, a local lesion host, and wheat, a systemic host, were used. A mixture of Japanese RNA1 transcripts and US RNA2 transcripts caused formation of local lesions on C. quinoa leaves and systemic infection to wheat plants. The nucleotide sequence of the progeny viral RNA2 was identical to that of the US RNA2. The reciprocal combination was not infectious to either host. These results confirm that the Japanese and US SBWMV are genetically distantly related strains belonging to a single species.

Evolutionary relationships and systematics of the alphaviruses.

Partial E1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus Alphavirus. Tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the Old or the New World, with at least two transoceanic introductions to account for their current distribution. The time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indicated that the nucleotide substitution rate varies considerably across sites within the genome. While most trees showed evolutionary relationships consistent with current antigenic complexes and species, several changes to the current classification are proposed. The recently identified fish alphaviruses salmon pancreas disease virus and sleeping disease virus appear to be variants or subtypes of a new alphavirus species. Southern elephant seal virus is also a new alphavirus distantly related to all of the others analyzed. Tonate virus and Venezuelan equine encephalitis virus strain 78V3531 also appear to be distinct alphavirus species based on genetic, antigenic, and ecological criteria. Trocara virus, isolated from mosquitoes in Brazil and Peru, also represents a new species and probably a new alphavirus complex.

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants.

A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUUUAG potential shifty-heptanucleotide at the end of the 5'-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient--1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5'-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3'-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known.

Construction of full-length cDNA clones to Soil-borne wheat mosaic virus RNA1 and RNA2, from which infectious RNAs are transcribed In vitro: virion formation and systemic infection without expression of the N-terminal and C-terminal extensions to the capsid protein.

The 19-kDa capsid protein (CP) of Soil-borne wheat mosaic furovirus (SBWMV) is encoded in the 5'-terminal region of RNA2. In addition to CP, two CP-related proteins are translated from SBWMV RNA2: (1) a 24-kDa protein (N-CP) with an N-terminal 40-amino-acid extension initiated at an upstream in-frame CUG codon; and (2) an 83-kDa protein (CP-RT) with an about 580-amino-acid, C-terminal extension by partial translational readthrough at the UGA termination codon at the end of the CP gene. We examined requirements for N-CP and CP-RT on virion formation and systemic infection in wheat plants using full-length cDNA clones, from which infectious RNA can be transcribed in vitro. RNA2 mutants, which could not synthesize N-CP, CP-RT, or either infected wheat plants systemically in combination with the wild-type RNA1 transcripts, produced rod-shaped virus particles in uninoculated upper leaves. Original mutations which abolished translation of N-CP and CP-RT were confirmed on RNA2 extracted from purified virus from the upper leaves by nucleotide sequence analysis. These results indicate that neither N-terminal nor C-terminal extensions to the CP are required for virion formation and systemic infection of SBWMV in wheat plants.

Suppressor mutations that allow sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis.

The alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30 degrees C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40 degrees C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40 degrees C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis.

Genome structure of Sagiyama virus and its relatedness to other alphaviruses.

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.

Similarity and divergence among viruses in the genus Furovirus.

Nucleotide sequences of RNAs 1 and 2 of a Japanese strain of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, and sorghum chlorotic spot virus (SCSV) were determined from cloned cDNA. The relationship among the Japanese and US strains of SBWMV, SCSV, oat golden stripe virus (OGSV), and recently proposed Chinese wheat mosaic and European wheat mosaic viruses (CWMV and EWMV) were examined at the nucleotide and amino acid levels. Pairwise comparisons of genome-encoded proteins among the six viruses showed that the US strains of SBWMV and CWMV were the most closely related pair in RNA 1 and the Japanese strains of SBWMV and EWMV were most closely related in RNA 2. SCSV was most distantly related to the other five viruses. Phylogenetic analysis indicated that there may have been an ancient reassortment between RNAs 1 and 2 of the four wheat-infecting viruses and OGSV, while SCSV was shown to have separated from the rest before the other five viruses diverged. The fact that CWMV and EWMV have almost identical biological properties as well as the sequence similarities to the two strains of SBWMV suggests that they be regarded as strains of SBWMV, considering that SBWMV consists of genetically diverged strains. OGSV and SCSV are distinct in biological properties in addition to genetic divergence in the genus Furovirus.

Comparison of nucleotide sequences between northern and southern philippine isolates of rice grassy stunt virus indicates occurrence of natural genetic reassortment.

Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia [corrected]. We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5' untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors.

Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase.

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.

Non-AUG translation initiation in a plant RNA virus: a forty-amino-acid extension is added to the N terminus of the soil-borne wheat mosaic virus capsid protein.

RNA 2 of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, encodes a protein previously hypothesized to be initiated at an in-frame non-AUG codon upstream of the AUG initiation codon (nucleotide positions 334 to 336) for the 19-kDa capsid protein. Site-directed mutagenesis and in vitro transcription and translation analysis indicated that CUG (nucleotides 214 to 216) is the initiation codon for a protein with a calculated molecular mass of 25 kDa composed of a 40-amino-acid extension to the N terminus of the 19-kDa capsid protein. A stable deletion mutant, which was isolated after extensive passages of a wild-type SBWMV, contained a mixture of two deleted RNA 2's, only one of which coded for the 25-kDa protein. The amino acid sequence of the N-terminal extension was moderately conserved and the CUG initiation codon was preserved among three SBWMV isolates from Japan and the United States. This amino acid sequence conservation, as well as the retention of expression of the 25-kDa protein in the stable deletion mutant, suggests that the 25-kDa protein is functional in the life cycle of SBWMV. This is the first report of a non-AUG translation initiation in a plant RNA virus genome.

Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses.

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.

Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis.

Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees C and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.

Complete nucleotide sequence and organization of the bipartite RNA genome of soil-borne wheat mosaic virus.

The complete nucleotide sequences of RNAs 1 and 2 of soil-borne wheat mosaic virus (SBWMV), type member of the furovirus group, were determined. RNA 1 is 7099 nucleotides (nt) and encodes a 150-kDa protein from the 5' end region, the UGA termination codon of which can be partially read through to produce a 209-kDa protein, and a 37-kDa protein in the 3' end region. The C-terminal region of the 150-kDa protein contains an NTP-binding helicase motif and the readthrough region, an RNA polymerase motif, indicating that these two overlapping proteins may form an RNA replication complex similar to those of tobamo- and tobraviruses. The 37-kDa protein has sequence similarity with the cell-to-cell transport protein of dianthoviruses. RNA 2 is 3593 nt and, from the 5' end region, encodes the 19-kDa capsid protein, whose UGA termination codon can be partially suppressed to produce an 84-kDa readthrough protein and, at the 3' proximity, a 19-kDa protein which is rich in cysteine residues. The 28K (kilodaltons, as estimated by SDS-PAGE) protein, previously considered as another capsid readthrough product, is apparently initiated at an in-frame non-AUG codon upstream from the capsid protein gene. In both RNAs 1 and 2, the 5' terminus is capped, and the 3' untranslated region possibly forms internal consecutive pseudoknots as found in tobamovirus RNA as well as a terminal tRNA-like structure similar to tymovirus RNA. An amino acid sequence comparison of RNA replicase genes indicates that, phylogenetically, SBWMV belongs to a cluster formed by tobamo-, tobra-, and hordeiviruses. Differences in the 3' end structure and in the cell-to-cell movement protein, and the distant phylogeny of the RNA replicase genes of SBWMV and beet necrotic yellow vein virus, suggest that the furoviruses should be divided into at least two groups.

Structure of the Ockelbo virus genome and its relationship to other Sindbis viruses.

Ockelbo virus was first isolated in 1982 in Sweden. It is the causal agent of disease in humans characterized by arthritis, rash, and fever and is antigenically very closely related to Sindbis virus. We have determined the nucleotide and translated amino acid sequences of the prototype Ockelbo virus isolate (82-5) to determine the relatedness of Ockelbo virus to Sindbis virus at the genomic level and clarify the taxonomic position of Ockelbo virus within the alphavirus genus. The numbers of nucleotides and of translated amino acids in each region of the Ockelbo virus genome were exactly the same as those for the prototype AR339 strain of Sindbis virus except for three small deletions and insertions in the C-terminal half of nsP3 and for three single nucleotide insertions and deletions in the 3' untranslated region. Overall there were 672 nucleotide differences (5.7% divergence), resulting in 97 amino acid changes (2.6% divergence), between the two viruses: more than 85% of the nucleotide changes were silent. Only the C-terminal domain of nsP3 and the E2 glycoprotein showed a higher degree of amino acid substitution than the overall average. The former domain is not conserved among alphaviruses, and the latter is primarily responsible for antigenic variation. Sequence analysis of 420 nucleotides at the 3' end of a number of other Sindbis-like alphaviruses, including Karelian fever virus and South African, Indian, and Australian isolates of Sindbis virus, demonstrated that Ockelbo virus is more closely related to South African strains of Sindbis virus than it is to the prototypic Egyptian AR339 strain. Thus the South African strains, which have caused epidemic disease in humans, may have been introduced into Northern Europe by man or by migratory birds to establish Ockelbo disease there. The Indian and Australian strains form a distinct branch of the evolutionary tree and differ from prototypic AR339 Sindbis virus in 17% of the nucleotides sequenced.

Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo.

The non-structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2-containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2-containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.

Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123.

The cleavage between nsP1 and nsP2 and that between nsP2 and nsP3 in the Sindbis virus nonstructural polyproteins was studied with respect to order of processing and enzyme-substrate relationships, using site-specific mutants in which the cleavage sites had been altered. The penultimate Gly in nsP1 or nsP2 or both was substituted by Ala, Val, or Glu, and processing was studied in vitro. Substitution with Ala resulted in partial cleavage whereas substitution with Val or Glu totally abolished cleavage at the mutagenized site. Abolishment of cleavage at the nsP2/nsP3 site did not affect processing at the nsP1/nsP2 site in the precursor polyprotein P123, and nsP1 and P23 were produced. When cleavage at the nsP1/nsP2 site was abolished, however, processing at the nsP2/nsP3 site was also prevented and P123 accumulated. To investigate why cleavage at the nsP1/nsP2 site should be required for cleavage at the nsP2/nsP3 site, the mutagenized polypeptides were used as enzymes in trans-cleavage experiments. We found that P123 can cleave the nsP1/nsP2 site but not the nsP2/nsP3 site, whereas P23 can cleave the nsP2/nsP3 site very efficiently. Thus, cleavage at the nsP1/nsP2 site by P123 is required to produce an enzyme capable of cleaving the nsP2/nsP3 site. Release of nsP4 from P1234 appears to be independent of the other cleavages and occurs primarily immediately after translation. These mutations were also transferred into a full-length cDNA clone of Sindbis virus and virus was recovered. Mutants defective in the cleavage of the nsP2/nsP3 site were temperature sensitive, growing at a slightly reduced rate compared to wild-type virus at 30 degrees but growing poorly at 40 degrees. Mutants defective in the cleavage of both the nsP1/nsP2 site and the nsP2/nsP3 site were viable but grew poorly compared with wild-type at any temperature.