Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex

Properties of non-structural protein 1 of Semliki Forest virus and its interference with virus replication

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5–10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis

Semliki Forest viirusel põhinevad vektorid ja rakuliinid alfa- ja hepatsiviiruste replikatsiooni ja interaktsioonide uurimisel

Semliki Forest virus (SFV; genus Alphavirus) has provided basic knowledge on the molecular biology of alpha­viruses, but also elucidated several general viral and cellular processes. The simple genomic organization of alphaviruses and their efficient propa­gation in cell culture have made alphavirus-based vectors promising tools for both DNA and RNA vaccination as well as for anti-cancer therapy. For these same reasons, alphavirus-based vectors can be potent systems for biotechno­logical applications. This thesis focuses on the properties of the non-structural protein 1 (nsP1) of SFV and the development and utility of novel SFV-based vector systems. Studies of the properties of SFV nsP1 and its non-palmitoylated variants revealed that mutations in the nsP1 palmitoylation site significantly reduce the infectivity of SFV genomes or SFV-based replicons. During infection of cell cultures, positively selected viruses with compensatory mutations emerged. It was found that nsP1, carrying the initial mutation, had an altered subcellular localization and was not able to interact with the polymerase subunit of the alphavirus replicase complex. The introduction of respective compensatory mutations restored, at least in part, these properties. As a part of studies, novel SFV replicon vectors expressing anti-apoptotic bcl-2 were constructed and analyzed. Although bcl-2 was expressed at high levels, its presence did not prolong the expression of a marker gene nor rescue the infected cells from virus-induced death. At the same time, these bicistronic vectors were found to be suitable for synchronic and transient expression of two genes of interest. Additio­nally, SFV-based vectors were successfully used to demonstrate an interaction between non-structural proteins of hepatitis C virus (HCV). Semliki Forest viirus (SFV) kuulub perekonda Alphavirus sugukonnas Togavi­ridae. Lihtsa ülesehituse ja efektiivse paljunemise tõttu on alfa­viirused olnud oluliseks töövahendiks erinevate viiruslike ning rakuliste protses­side kirjeldamisel; samas on alfaviirustel-põhinevad vektorid palju­tõotavad geeni- või kasvajavastases teraapias ning biotehnoloogias. Käesoleva töö eesmärkideks oli uurida SFV mittestruktuurse valgu nsP1 ning tema erinevate mittepalmitüleeritud vormide mõju viirusnakkusele ning peremeesrakule. Samuti uuriti võimalust takistada anti-apoptootilise valgu bcl-2 ekspressiooni abil SFV poolt põhjustatud apoptoosi ning kasutati SFV-l põhi­nevaid vektoreid C-hepatiidi viiruse proteaaside interaktsioonide kirjelda­miseks. Uurimine näitas, et palmitüleerimist takistavate mutatsioonide sisseviimine nsP1 kodeerivasse alasse vähendas järsult viiruse paljunemise efektiivsust. Samas taastasid replikatsiooni käigus tekkinud ja selekteerunud kompenseeriva toimega mutatsioonid RNA-de nakkuslikkuse ja viiruse paljunemise efektiivsuse. Leiti, et mitte-palmitüleeritud mutantide funktsionaalset defekti ei põhjustanud palmitüleerimise kui sellise puudumine, vaid hoopis nsP1 mutantsete vormide võimetus seonduda SFV polümeraasi katalüütilise subühikuga. Uudsete bitsistroonsete SFV replikon-vektorite abil leiti, et vähemalt BHK-21 rakkudes ei takista bcl-2 ekspressioon SFV indutseeritud apoptoosi. Samas on konstrueeritud vektorid efektiivseteks töövahenditeks juhtudel kui on vaja transientselt ja sama-aegselt ekspresseerida kahte uuritavat valku. SFV-vektorite kasutamisel näidati, et C-hepatiidi viiruse proteaasid NS2 ja NS3 moodustavad omavahel kompleksi. Samuti leiti, et kompleksi moodustumine ei sõltu peremeesraku tüübist ega sellest, kas vastavad valgud on ekspresseeritud paarikaupa või ühise eelvalgu kujul

Lysosomes and endosomes are enriched in IFN-inducing RNA generated by mutant viral replicase.

<p>(A) COP-5 cells were transfected with pRep-RDR or pRep-RDR/GAA plasmid DNA. After 48 hr, post-nuclear supernatant (PNS) was prepared and fractionated by flotation in a sucrose step gradient. Subsequently, naïve COP-5 cells were transfected in equimolar amounts with RNAs extracted from each fraction, and IFN-β levels were measured by ELISA. Lys+LE, lysosomes and late endosomes; EE, early endosomes; Gol+ER, Golgi complex and endoplasmic reticulum; Cyt, cytosol. (B) COP-5 cells were transfected with pRep, pRep-RDR, or pRep-RDR/GAA plasmid DNA. After 48 hr, the cells were fixed, permeabilized and stained with anti-nsP1 and anti-dsRNA antibodies. The cells nuclei were counterstained with 4,6-diamino-2-phenylindoldihydrochloride. (C) RD cells were transfected with pRep-RDR plasmid DNA. After 48 hr, the cells were fixed, permeabilized and stained with different combinations of anti-nsP1, anti-dsRNA, and anti-LAMP2 antibodies. The nuclei were counterstained as described in (B). Error bars (A) represent the standard deviation of two experiments. All images (B and C) are representative of three independent experiments.</p

Mutant SFV Replicase triggers IFN-β in a RIG-I- and MDA-5-dependent manner.

<p>(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinations thereof. After 48 hr, cells were transfected with poly(I:C) dsRNA, and after 24 hr, the amount of secreted IFN-β was measured by ELISA (upper panel). The efficiency of RLR protein knockdown was assessed by immunoblot assay (lower panel). Cells lysates were separated by SDS-PAGE and immunoblotted with different antibodies. Neg. ctrl., negative control non-targeting siRNA; mock, transfection without siRNA. (B) COP-5 cells were transfected with siRNAs as described in (A). After 48 hr, cells were transfected with pRep-RDR plasmid DNA, and after 48 hr, the amount of secreted IFN-β was measured as described in (A) (upper panel). The RLR knockdown efficiency (lower panel) was assessed as described in (A). Expression of SFV replicase was analyzed using antibodies against nsP4 and nsP2; ACTIN was used as loading control. (C and D) MEFs were transfected with siRNAs as described in (A). After 48 hr, cells were infected with SFV4-Rluc-RDR at different MOIs. After an additional 12 hr, the amount of IFN-β was measured (C) and cell lysates were prepared for the Renilla luciferase reporter assay (D). Immunoblots (A and B) and panels (C and D) are representative examples of two independent experiments. Error bars (A and B) represent the standard deviation of three experiments. ***p<0.001 (t-test).</p

Model of mutant SFV replication restriction in fibroblasts.

<p>Transcription of the host cell RNA by the SFV replicase leads to generation of 5′-ppp dsRNA, which is detected by RLRs (RIG-I and MDA-5). Subsequently, type I IFN is induced and secreted. Released type I IFN activates transcription of interferon-stimulated genes (ISG), which trigger viral replication restriction by degrading SFV genomes.</p

IFN-β induction during SFV infection can not be exclusively explained by the presence of virus-specific RNAs.

<p>(A) Polyadenylated (polyA+) and non-polyadenylated (polyA−) RNA fractions obtained by oligo(dT)-affinity chromatography fractionation from total RNA extracted from mock-, SFV4-Rluc-, and SFV4-Rluc-RDR-infected MEF cells. RNA samples (500 ng/lane) were resolved using electrophoresis on a non-denaturing 0.8% agarose gel and visualized by ethidium bromide staining. Quantitation of the amounts of 42S (±) dsRNA was performed with ImageJ; results are shown at the bottom of lanes 3 and 4, 5 and 6. pA+, polyadenylated RNA fraction; pA−, non-polyadenylated RNA fraction; RNA M1, RNA marker 1 (top – 6, 4, 3, 2, 1.5, 1.0, 0.5, 0.2 – bottom, Knts); RNA M2, RNA marker 2 (top – 1, 0.8, 0.6, 0.4, 0.3, 0.2, 0.1 – bottom, Knts); DNA M, DNA marker (top – 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1.0, 0.75, 0.5, 0.25 – bottom, Kbps). (B) PolyA+ and polyA− RNA fractions shown in (A) were resolved using electrophoresis on denaturing formaldehyde 0.8% agarose gel and transferred to nylon membrane. Subsequently, northern hybridization analysis with fragmented DIG-labeled SFV4-Rluc RNA probe (200 ng/ml) was performed. Equal amounts of RNA (30 ng) were loaded into each lane. (C) The increasing amounts of RNAs shown in (A) were UV-treated and transfected into COP-5 cells, while the total RNA amount was kept constant with “stuffer” RNA (total RNA from naïve COP-5 cells). At 12 hr post transfection, the amount of IFN-β was determined in the cell culture medium by ELISA. ND, not detectable; h.p.t., hours post transfection. (D) The non-polyadenylated RNA fractions shown in (A) were size-fractionated on silica-columns, and 1/10 of the volume of RNA was analyzed on a non-denaturing 0.8% agarose gel; RNAs were visualized with ethidium bromide staining. (E) Large RNAs (>200 nt) or small RNAs (<200 nt) shown in (D) were transfected into COP-5 cells, and IFN-β levels were measured by ELISA after 12 hr. ND, not detectable; h.p.t., hours post transfection; 10×RNA <200, 10-fold molar excess of small RNAs compared with large RNAs. Error bars (C and E) represent the standard deviation of three independent experiments.</p

SFV replicase transcribes host cell RNA, triggering IFN-β induction during SFV infection.

<p>(A) Equal amounts (1 µg) of polyA− RNA fractions obtained by oligo(dT)-affinity chromatography fractionation from total RNA extracted from mock-, SFV4-Rluc-, and SFV4-Rluc-RDR-infected MEF cells were either treated (right) or not treated (left) with RNA 5′ polyphosphatase. Subsequently, RNAs were either denatured (black bars) or not denatured (white bars) and transfected into COP-5 cells. At 12 hr post transfection, the amount of IFN-β was determined in the cell culture medium by ELISA. ND, not detectable; h.p.t., hours post transfection. (B) SFV4-Rluc polyA− RNA fraction was resolved by performing electrophoresis on a native low melting 0.8% agarose gel and RNA species corresponding to viral dsRNA RF 42S (±), viral ssRNA 26S (+)/28S rRNA, 18S rRNA, and 5S rRNA were purified. Subsequently, obtained RNA species were mock-, RNase T1-, and alkaline phosphatase (AP)-treated and transfected into COP-5 cells, while the total RNA amount was kept constant with “stuffer” RNA (total RNA from naïve MEF cells). At 12 hr post transfection, the amount of IFN-β was determined in the cell culture medium by ELISA. h.p.t., hours post transfection. (C) Schematic of the strategy used to tag and amplify RNA fragments generated by SFV replicase (top). Obtained PCR products were resolved using electrophoresis on the 2% agarose gel (in tris-borate-edta buffer) (bottom). DNA M, DNA marker (top – 700, 500, 400, 300, 200, 150, 100, 75, 50, 25 – bottom, bps). (D) Unique cloned RNA identified using the strategy, shown in (C). Schematic representation of the stem-loop RNA is based on the sequence alignment (BLASTN 2.2.28+ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003610#ppat.1003610-Altschul1" target="_blank">[91]</a>) of mouse ASncmtRNA-1 and human ASncmtRNA-2, for the latter such structure was experimentally confirmed <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003610#ppat.1003610-Burzio1" target="_blank">[63]</a>. (A) and (B) are representative of two independent experiments.</p

Properties of IFN-inducing RNA generated by SFV replicase.

<p>(A) COP-5 cells were transfected with either pRep-RDR, pRep-RDR/GAA or poly(I:C) or were mock-transfected. After 48 hr, the cells were lysed and the total RNA was size-fractionated on a silica column. The two resulting fractions, containing either large RNAs (>200 nt) or small RNAs (<200 nt), were transfected into MEFs, and IFN-β levels were measured by ELISA after 24 hr. Mock, transfection without DNA or RNA; ND, not detectable; 10×RNA <200, 10-fold molar excess of small RNAs as compared to large RNAs. (B) COP-5 cells transfected with pRep-RDR were lysed at 48 hr post transfection. The total RNA was extracted and fractionated into polyadenylated and non-polyadenylated RNA fractions using oligo(dT)-affinity chromatography. Increasing amounts of the obtained RNA fractions were transfected into COP-5 cells, while the total RNA amount was kept constant with “stuffer” RNA (naïve COP-5 total RNA). At 24 hr after transfection, the amount of IFN-β was determined in the cell culture medium by ELISA. polyA+, polyadenylated; polyA−, non-polyadenylated. (C) Upper panel: dsRNA probes were treated with various amounts of the indicated RNase, separated on agarose gel, and then visualized by ethidium bromide staining. Lower panel: Total RNA was extracted from pRep-RDR-transfected cells and digested with various amounts of the indicated RNase or treated with DNase I or alkaline phosphatase (AP) and analyzed as described in (C, upper panel). Mock, no enzyme added. (D) RNAs (C, lower panel) were transfected into COP-5 cells, and ELISA was used to measure the amount of secreted IFN-β after 24 hr. Error bars (A, B, and D) represent the standard deviation of two experiments.</p