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The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production.

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues (648)RRR(650) with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3'-truncated pgRNA lacking 3' direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.

Alphaviruses are small enveloped viruses that have been used extensively as model enveloped viruses. During infection, virus particles are taken up into endosomes, where a low pH activates the viral fusion protein, E1. Fusion of the viral and the endosomal membranes releases the viral core into the cytoplasm where cores are disassembled by interaction with 60S ribosomal subunits. Recently, we have shown that in vitro this disassembly is strongly stimulated by low pH. We have proposed that after entry of the core into the cytoplasm, the viral membrane proteins that have been transferred to the endosomal membrane form an ion-permeable pore in the endosome. The resulting flow of protons from the endosome into the cytoplasm through this pore could generate a low-pH environment for core disassembly in vivo. Here we report two types of analysis aimed at the identification of such pores. First, the release of [3H]choline from the interior of liposomes was analysed in the presence of virus particles and viral proteins. Secondly, cells were infected with Sindbis or Semliki Forest alphaviruses at the plasma membrane and the possible generation of ion-permeable pores during this process was analysed by whole-cell voltage clamp analysis of the membrane current. The results obtained indicated that the proposed pores are in fact generated and allowed us to identify the formation of individual pores. Available evidence indicates that the alphavirus E1 protein probably forms these pores. Proteins homologous to the alphavirus E1 protein are present in flaviviruses and hepatitis C virus.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2.6x10(5) to 1:1x10(6)). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII+RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 degrees C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15,000 g (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.

The nucleolar accumulation of Semliki Forest Virus (SFV) C protein was examined as a function of intact microtubules, intact microfilaments and accessible intermediate filaments. The cytoskeletal components do not seem to play a role in directing C protein to the nucleolus but nucleolar accumulation is energy-dependent and saturable. This suggests the involvement of some receptor- (or chaperon-) interaction.

The processing and stability of the non-structural (ns) proteins of Semliki Forest virus were studied in vivo. Virus-specific proteins from infected cells were identified by immunoprecipitation with monospecific antisera. The complete ns precursor, P1234, translated within 7 to 9 min of the start of translation, was processed before the completion of translation into P123 and nsP4. Pulse-chase experiments showed that the mature ns proteins were relatively stable for at least 2 h. Interestingly, the decrease in the amount of the P34 precursor during chase is accompanied by an increase only in the amount of nsP3, which could explain the observed lower amount of nsP4 in infected cells. In cells infected with SFV RNA- mutants ts4 and ts6 maintained at the restrictive temperature, nsP4 but no nsP1, nsP2 or nsP3 accumulated in addition to the ns precursor proteins P1234, P123, P12 and P34. Translation in vitro of mRNAs from a cDNA clone, encoding P1234 with a deletion in the carboxy-terminal proteinase domain of nsP2, did not yield nsP1, nsP2 or nsP3 but only nsP4, indicating that cleavage at the nsP3/4 sites is independent. Evidently, nsP4 is produced by a nascent cleavage of the growing P1234, catalysed by its own proteinase activity; the proteolytic cleavages at the nsP1/2 and nsP2/3 sites are catalysed by the proteinase moiety of nsP2.

Two previously undescribed stable polypeptides (referred to as nsp 90 and nsp 63) appear in mammalian and avian cells infected with Semliki Forest virus. They are distinguishable from the virus structural proteins and their known precursors by their molecular weights and tryptic peptide maps, and are identical in size to two polypeptides found in purified preparations of virus-specific RNA polymerase. Data from pulse-chase experiments and from the use of inhibitors of proteolytic cleavage indicate that nsp 90 and nsp 63 are synthesized via a series of post-translational cleavages from three larger polypeptides, p200, p184 and p150. The labelling kinetics after synchronous initiation of protein synthesis are also consistent with the synthesis of nsp 90 and nsp 63 from a common initiation site, and show that nsp 63 is located close to this site. It is concluded that nsp 90 and nsp 63 are components of the virus-specific RNA polymerase, and are synthesized via a post-translational cleavage scheme entirely separate from that leading to the synthesis of the virus structural proteins.

Purified defective-interfering (DI) particles of Semliki Forest virus are unable to carry out any of the steps in virus multiplication except uncoating. Cells co-infected with DI particles and standard virus contain several virus-specified RNA species (DI particle-specific species) absent from cells infected with standard virus alone. Moreover, synthesis of all the virus-specified components distinctive of standard virus-infected cells is reduced. The DI particle-specific RNA species comprise two poly A-containing single-stranded RNAs (DIss1 and DIss2), identical to those found in purified DI particles, two double-stranded RNA'S (RFs) and a new size class of replicative intermediate (RI). Hybridization experiments showed that the nucleotide sequences of DIss1 and DIss2 (i) are present in the 42S genome of standard virus but absent from the 26S RNA- the RNA from standard virus-infected cells which encodes the structural proteins of the virion (Clegg & Kennedy, 1975 a) and (ii) are complementary to the negative strands of the DI paritcle-specific RFs and RI. Oligonucleotide fingerprinting revealed extensive nucleotide sequence homology between DIssI and DIss2. Analysis of the mRNA complement of standard virus-infected, co-infected and uninfected cells strongly indicated that neither DIss1 nor DIss2 can serve as a functional messenger RNA. From these studies we propose a mechanism for the multiplication of and interference by DI particles of Semliki Forest virus.

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.

RAN synthesis induced by a temperature-sensitive RNA negative mutant, ts-4, of Semliki Forest virus was studied both at the permissive (28 degrees C) and restrictive temperature (39 degrees C). At 28 degrees C ts-4 directed RNA synthesis was qualitatively similar to the wild type but overall synthesis remained somewhat lower. Only about 3% of RNA was synthesized compared to the wild type virus when ts-4 was maintained at 39 degrees C throughout the infection. The sedimenting radioactivity was almost exclusively in 42S RNA. When ts-4 infected cultures were shifted from 28 to 39 degrees C at 5 h post-infection, the synthesis of 26S RNA was shut off while 42S RNA synthesis continued. The synthesis of 26S RNA started again even in the presence of cycloheximide if the cultures were back to 28 degrees C. Our results suggest that 26S RNA synthesis is controlled by a virus-specific protein, the function of which is not required for the synthesis of 42S RNA, In ts-4 this protein is denatured at 39 degrees C and becomes non-functional, but renatues and becomes functional upon shift down to 28 degreesC.

The synthesis of Semliki Forest virus nucleocapsid in infected cells was studied by labelling the virus RNAs with 3H-uridine for different periods at various phases of infection. Short pulses (10 to 20 min) revealed the accumulation of 42S RNA in a ribonucleoprotein which sedimented at about 90S (90S RNP) and contained only small amounts of capsid protein. Only after longer pulses was the labelled 42S RNA found in the virus nucleocapsid, suggesting that the 90S RNP may be its precursor. The life time of the 90S RNP was long in the early phases of infection and short in the late phases, reflecting the increased rate of assembly of the nucleocapsid during infection. The 90S RNP was the only 42S RNA containing RNP found in cells infected with temperature sensitive mutants deficient in nucleocapsid formation or wild type infected cells treated with cycloheximide to inhibit nucleocapsid assembly.

In Semliki Forest virus (SFV)-infected cells, all structural proteins are translated from a 26S mRNA using a single initiation site. The capsid protein which is made first is released into the cytoplasm whereas the two membrane proteins, p62 (the precursor for E2 and E3) and E1, are inserted into the rough endoplasmic reticulum membrane. Based on gradient centrifugation and cross-linking studies, it can be seen that the p62 and E1 polypeptides form a complex immediately after synthesis and migrate to the plasma membrane in the form of a p62-E1 complex. The processing of p62 to E2 and E3 is first seen 25 to 30 min after a 10 min pulse of radioactive amino acids. This cleavage can be inhibited by addition of antisera specific for E1 and E3, thus supporting the view that, as in the case of the related Sindbis virus, this cleavage occurs on the external face of the plasma membrane. Proteolytic digestion of crude vesicle preparations derived from plasma membranes, combined with peptide mapping, indicate that the carboxy-terminal end of E2 spans the cell plasma membrane, there being a portion of mol. wt about 3000 located towards the cytosol.

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

Three replicative forms of RNA (RF I, RF II, and RF III) have been isolated from BHK cells infected with Similiki Forest virus. Using analytical and rate-zonal sedimentation the mol. wt. of these replicative forms were estimated to be 8-5 times 10(6), 5-5 times 10(6) and 3-1 times 10(6) respectively. After continuous labelling from 1 to 6 h post-infection, RFI constituted more than 80% of the total replicative forms. Competition hybridization experiments showed that one strand of RFI was 42S RNA which had opposite (negative) polarity to that found in the virus particle. The positive strand of RFI was 42S RNA. The negative strand of replicative intermediate (RI) was also found to be 42S RNA. No evidence was found for an RI with a 26S negative strand. RFI was shown to contain non-hydrogen bounded poly A at or near the 3' end of the component 42S positive strand. Isolation and analysis of the poly A tract from RFI on an acrylamide gel showed it to be of essentially the same average size as the poly A tract from virus particle RNA. About 30% of the RI molecules contained non-hydrogen bonded poly A. No poly U was detected in either RFI or RI. The kinetics of positive and negative strand synthesis were investigated during virus multiplication. These experiments showed that the rate of negative strand synthesis reaches a maximum 2 1/2 post-infection and thereafter rapidly falls. The rate of positive strand synthesis increases rapidly up to 3 h post-infection and then remains constant for a further 3 to 4 h.

When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 degrees C) and shifted to the restrictive temperature (39 degrees C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al. 1977) and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 degrees C, indicating that no new active RNA polymerase is formed at 39 degrees C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 220 000 (ns220) was demonstrated in ts-4 infected cultures shifted to 39 degrees C. NS220 was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns220 are located near the initiation site at the 5'-end of the 42S RNA. The viral specificity of ns220 was shown by its disappearance after a shift to 28 degrees C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 degrees C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation.