Sirkka Keränen

IDENTIFICATION OF A PRECURSOR FOR ONE OF THE SEMLIKI FOREST VIRUS MEMBRANE PROTEINS

We are studying the envelope of Semliki forest virus (SFV) as a model for membrane structure and biogenesis. Semliki forest virus and other group A arboviruses acquire their envelopes by budding through the host cell plasma membrane [ 1] . The viral envelope resembles the host cell plasma membrane in its lipid composition [2] , but has a much simpler protein composition: one protein with an apparent molecular weight of about 50,000 has been found in the membrane [3]. In Sindbis virus this protein has recently been split into two bands (Er and Ez) using discontinuous sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis [4]. Both of these polypeptides contain carbohydrate. Studies on SFV protein formation are facilitated by the fact that virus infection effectively shuts off host cell protein synthesis [5]. Attempts to find out whether the structural polypeptides of the virus are translated as primary gene products or in precursor form(s) have so far been inconclusive [6-91. Our previous studies have suggested a precursor role for a non-structural glycopolypeptide (NSP68) which is found in extracts of infected cells [lo]. This polypeptide has an apparent molecular weight of 68,000 in 5% acrylamide SDS-gels. Here we show that SFV contains two envelope polypeptides, El and Ea , and that these have different primary structures. We also show that NSP68 is a precursor for the envelope polypeptide Ez, which is confirmed by the isolation of a temperaturesensitive mutant of SFV, in which precursor cleavage is blocked.

A sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis

Abstract The synthesis of Sindbis virus (SIN) minus-strand RNA which is utilized as a template for the synthesis of 49 S plus-strand RNA and the 26 S mRNA is normally short-lived and regulated. Minus-strand RNA synthesis ceases 3–4 hr after infection or quickly after the inhibition of protein synthesis; this is in contrast to plus-strand RNA synthesis which continues throughout the infectious cycle and is resistant to inhibition of protein synthesis. In cells infected with SIN HR ts24, a member of the A complementation group, the synthesis of minus-strand RNA ceased normally at 6−8 hr p.i. when the infection was at 28°, the permissive temperature. However, shifting SIN HR ts24-infected cells to the nonpermissive temperature, 39°, during the early phase of the infectious cycle resulted in the failure to turn off minus-strand transcription, the accumulation of replicative intermediates, and the loss of sensitivity of minus-strand RNA synthesis to inhibition of protein synthesis. Furthermore, shifting SIN HR ts24-infected cells to the nonpermissive temperature late in the infectious cycle, after minus-strand synthesis had ceased normally at 28°, resulted in the resumption of minus-strand synthesis, even in the absence of protein synthesis. Thus, SIN HR ts24 appears to have a temperature-sensitive lesion in a viral polypeptide that acts to turn off minus-strand synthesis and that may be responsible for the normal short-lived nature of the minus-strand polymerase.

Replication of the Genome of Alphaviruses

SUMMARY The genome of Semliki Forest virus (SFV) is 11 442 nucleotides with a 5′ cap-structure and a 3′ poly(A) tail of about 100 residues. The genome of the closely related Sindbis virus (SIN) is slightly longer (11 703 nucleotides). The parental RNA is first translated from the 5′ two thirds to yield; nsP1, nsP2, nsP3 and nsP4, which are cleaved from a polyprotein of 2431 amino acids (SFV). The parental genome is copied to a full-length minus strand with poly(U) at the 5′ end. The minus strand is used as template for the synthesis of 42 S RNA in membrane-bound replicative-intermediate (RI) structures. In addition to 42 S RNA, a 3′-coterminal subgenomic 26 S mRNA, coding for the structural proteins, is synthesized by internal initiation at the minus strand. Capping and methylation of both plus-strand RNAs occur concomitantly with their synthesis. Analysis of Sindbis virus temperature-sensitive RNA-negative mutants have shown that one complementation group (B) is specifically associated with the synthesis of minus strands. Another, group F, is involved in the polymerization step of both minus- and plus-strand 42 S RNA, and of the 26 S mRNA. The synthesis of minus strands is normally dependent on protein synthesis. There is a shut off of the minus-strand RNA synthesis at about 3 h post-infection. This is apparently regulated by a virus-specific protein, represented by the complementation group A. The same protein is involved in the regulation of the initiation of 26 S RNA together with a component represented by group G mutants. Comparative analysis of SFV and SIN RNAs and DI RNAs of both viruses suggests that perhaps only 19 nucleotides from the 3′ end and about 150 nucleotides from the 5′ end are needed for replication of the alphavirus RNAs. In some SIN DI RNAs the proposed secondary structure at the 5′ end is replaced by a cellular tRNAASP suggesting that the secondary structure rather than nucleotide sequence is sufficient for the recognition by the viral polymerase. Even when the primary structure of the four non-structural proteins of both SFV and SIN is known, the correlation of the genetic data with the individual proteins has not yet been possible.

Extreme ends of the genome are conserved and rearranged in the defective interfering RNAs of Semliki Forest virus.

Abstract We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb † ) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome. The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified.

Semliki forest virus mutants with temperature-sensitive transport defect of envelope proteins

Abstract Seven RNA-positive temperature-sensitive mutants of Semliki Forest virus were studied to find mutants with a defect in the transport of envelope glycoproteins to the host cell plasma membrane. Surface immunofluorescence of infected cells was recorded at the restrictive temperature (39°) and after shift to the permissive temperature (28°) carried out in the presence or absence of protein synthesis inhibitors. Preformed envelope proteins of two mutants, ts-1 and ts-7, were found at the plasma membrane only after shift to the permissive temperature. Staining of intracellular antigens after mild detergent treatment of ts-1 and ts-7 infected cells showed that at 39° the envelope proteins had reached th Golgi apparatus. Electron microscopy of ts-1 and ts-7 infected cells, maintained at 39°, showed that all the nucleocapsids were free in the cytoplasm. In ts-7, but not in ts-1, infected cells binding of nucleocapsids and virus budding started soon after the cultures had been shifted to the permissive temperature. First the budding was observed only at the Golgi and later also at the plasma membrane. Envelope proteins labeled at the restrictive temperature were efficiently incorporated into ts-7 but not into ts-1 virus particles, released after shift to 28°. We suggest that the viral glycoproteins have different “signals” for the transport and for the binding of nucleocapsid. In ts-7 both “signals” become functional after shift to 28°, whereas in ts-1 only the “transport signal” defect is reversible.

Efficient secretion of Bacillus amyloliquefaciens α-amylase cells by its own signal peptide from Saccharomyces cerevisiae host

Abstract The expression and secretion of Bacillus amyloliquefaciens α-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the α-amylase gene were constructed: the Bacillus signal sequence was either complete (YEpαal), partial (YEpαa2) or missing (YEpαa3). Secretion of α-amylase into the culture medium was obtained with the complete signal sequence only. The secreted α-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated α-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.

Multiple structurally related defective-interfering RNAs formed during undiluted passages of Semliki forest virus.

Abstract Semliki Forest virus (SFV) was transferred serially with undiluted inoculum for 24 successive passages in BHK cells and the virus-specific RNAs were isolated from infected cells and from virus particles released into the medium. The infectivity and hemagglutination titers started to drop after 4 serial passages. A new virus-specific intracellular RNA species sedimenting at 18 S appeared in the 4th passage and remained the dominant DI-RNA up to the 17th passage. The 18 S RNA population was incorporated very inefficiently into viral particles, but was replicated more rapidly than other viral RNAs. Another DI-RNA, sedimenting at 24 S, was found in virus particles starting at the 4th passage. The 24 S RNA replicated less efficiently but was incorporated in virus particles 3 to 5 times more efficiently than the 18 S RNAs. After 18 undiluted passages a switch in the distribution of intracellular RNAs resulted in the predominance of a 24 S RNA species. The early and late 24 S RNA species were found to be related but not identical when compared by ribonuclease Tl oligonucleotide mapping. The results indicated that the early 24 S RNA fingerprint closely resembled that of the 18 S DI-RNA population, whereas the late 24 S RNA had a lower complexity. A 33 S DI-RNA species isolated from virus particles at the 21st passage also had a simple T1 oligonucleotide pattern indistinguishable from that of the late 24 S RNA. These results suggest that the late 24 S DI-RNA molecules contain multiple repeated sequences similar to those recently found in the 18 S DI-RNA.

Semliki Forest Virus Replication Complex Capable of Synthesizing 42S and 26S Nascent RNA Chains

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

Characterization of GPI14/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae.

We report here identification and characterization of a mutation in the GPI14 gene, the yeast homologue of the mammalian PIG‐M that functions in the synthesis of the GPI moiety anchoring proteins to the plasma membrane. We show that the first putative transmembrane domain of Gpi14p is not essential for its function. Downregulation of GPI14 expression/reduced protein function due to an amino terminal deletion resulted in increased transcription and production of an endogenous and a heterologous secreted protein expressed from HSP150 and ADH1 promoter, respectively. In these cells, unfolded protein response was induced but was not responsible for the enhanced production of these proteins. A cell wall defect in the gpi14 mutant cells was suggested by cell aggregation phenotype, increased sensitivity to Calcofluor white, an increased release of Gas1p and total protein into the culture medium. In the gpi14 mutant cells, transcription of RLM1, a transcription factor participating in the cell wall integrity signalling pathway, was increased, and deletion of RLM1 resulted in a synthetic lethal phenotype with the gpi14 mutation. These results suggest that partial inactivation of Gpi14p causes defects in the cell wall structure and suggest that compromised GPI anchor synthesis results in enhanced protein production via the cell wall integrity signalling pathway. Copyright

Interference of wild type virus replication by an RNA negative temperature-sensitive mutant of Semliki Forest virus.

Abstract Interference of wild type Semliki Forest virus replication induced by an RNA negative temperature-sensitive mutant, ts -11, at the restrictive temperature was studied. This mutant induces a transient inhibition of RNA synthesis, resulting in delayed and reduced production of infectious virus. The interfering activity was retained in ts -11 preparations after three successive plaque-to-plaque clonings and purification of the mutant virus. The possibilities that interference was due to induction of interferon or RNase activity were excluded, nor was ts -11-directed RNA or protein synthesis required. It is suggested that the interference is caused by a structural component, RNA or protein, of the ts -11 virion, which inhibits the action of RNA polymerase.