Exploring RNA Virus Replication in Xenopus Oocytes Andrea V Gamarnik and Raul Andino

Summary

Microinjection of poliovirus RNA in Xenopus oocytes initiates a complete and authentic viral replication cycle that yields newly synthesized infectious virus. This system can be used to study the molecular mechanism of the different steps involved in virus replication. Interestingly, viral replication only occurs if poliovirus RNA is coinjected with factors present in HeLa extracts. We have determined that two HeLa cell factors are required for viral replication in oocytes, one involved in initiation of translation (polio translation factor) and the other in RNA synthesis. Thus, microinjection in oocytes provides a strategy to identify and further analyze the function of these host cell factors and to biochemically dissect the mechanism of initiation of poliovirus translation and RNA synthesis. Here, we review protocols, approaches, and potential issues that can be addressed using the oocyte system.

Key Words: Xenopus oocytes; RNA virus; viral RNA replication; negative strand RNA detection; viral protein synthesis; HeLa cell factors; infectious viral particles; IRES-dependent translation.

1. Introduction

Microinjection of viral RNAs into Xenopus oocytes constitutes a powerful system to study mechanistic aspects of viral replication. We have previously demonstrated that stage VI oocytes support replication of different members of the Picornaviridae family (1-4). These viruses followed a replication strategy common to other positivestrand RNA viruses (Fig. 1; ref. 5). After entry, the genomic RNA is released in the cytoplasm of the cell and functions as messenger RNA (mRNA) directing the synthesis of a large polypeptide, which is proteolytically processed to yield mature viral proteins.

The same RNA molecule is then amplified in a two-step process: first, its complementary negative strand is synthesized, and then the negative-strand RNA is used as a template to generate new molecules of positive-strand RNA (for review, see ref. 6). The synthesis of both negative- and positive-strand RNA is catalyzed by the viral RNA-dependent RNA polymerase. All these steps of replication, including the forma-

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

Fig. 1. Schematic representation of the replication of a positive-strand RNA virus. Infection starts with the binding of the virus to specific receptors on the cell surface. The viral RNA is uncoated and delivered into the cytoplasm. Viral RNA is translated to yield the structural and nonstructural viral proteins. Subsequently, cellular and viral proteins enable replication of the genomic RNA, which serves as a template to synthesize a complementary negative-strand RNA, which is employed to amplify the viral genome. Finally, the newly synthesized positive-strand RNA is packaged into newly synthesized virions.

Fig. 1. Schematic representation of the replication of a positive-strand RNA virus. Infection starts with the binding of the virus to specific receptors on the cell surface. The viral RNA is uncoated and delivered into the cytoplasm. Viral RNA is translated to yield the structural and nonstructural viral proteins. Subsequently, cellular and viral proteins enable replication of the genomic RNA, which serves as a template to synthesize a complementary negative-strand RNA, which is employed to amplify the viral genome. Finally, the newly synthesized positive-strand RNA is packaged into newly synthesized virions.

tion of new infectious particles, can be faithfully reproduced in the oocyte system. Infectious virus is detected as early as 6 h postinjection (100 PFU/oocyte), and the amount of virus increases exponentially, reaching a maximum of 5 x 105 PFU/oocyte after 15 h.

This chapter describes the methods and techniques routinely used in our laboratory to analyze each step of poliovirus replication and provides some practical suggestions to identify and characterize host factors involved in these processes. We believe that this methodology could be easily extrapolated to study the replication of other RNA viruses.

2. Materials

1. Collagenase type I, 250 U/mg (CLS 1; Worthington Biochemical Corp.).

2. Proteinase K, stock solution (2 mg/mL); keep at -20°C.

3. Actinomycin D, 5-mg/mL stock solution in ethanol. Store the stock solution in a foil-wrapped vial at -20°C (it is light sensitive).

4. Phenol/chloroform (1/1) buffer equilibrated to pH 6.7.

5. [35S] methionine 15 mCi/mL.

6. [a-32P] Guanosine 5'-triphosphate (GTP) 10 mCi/mL.

7. Buffer H (10X stock solution keep at -4°C): 100 mM HEPES, pH 7.9, 500 mM KCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), 5 mM Triton X-100.

8. Modified Barth's solution (MBS): 7.5 mM Tris-HCl at pH 7.6, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 8.2 mM MgSO4, 0.33 mM Ca(NO3)2, 0.4 mM CaCl2, 100 U/mL penicillin, 100 |lg/mL streptomycin. 2% Ficoll-400.

9. Buffer H: 10 mM HEPES at pH 7.9, 50 mM KCl, 2 mM EDTA, 0.5 mM PMSF, 1 mM DTT, 0.5 mM Triton X-100.

10. Hypotonic buffer: 20 mM HEPES at pH 7.4, 10 mM KCl, 1.5 mM Mg(CH3CO2)2, 2 mM DTT.

11. TENSK: 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate (SDS), 200 | g/mL proteinase K.

12. TSE buffer: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA.

14. Restriction enzymes.

15. T7 RNA polymerase.

16. SP6 RNA polymerase.

17. 10 mM Nucleotide triphosphate (NTP) mix.

19. Nitrocellulose membranes.

20. Electrophoresis equipment.

21. Dounce homogenizer.

22. HeLa S3 cells. 21. Tissue culture facilities.

3. Methods

3.1. Translation of Viral RNAs in Xenopus Oocytes

The first step of positive-strand RNA virus replication is the translation of the genomic RNA. Viral RNAs either resemble cellular mRNAs by having a cap structure at the 5' end or have different RNA elements, which initiate translation by cap-independent mechanisms (7). In the case of picornaviruses, the 5' untranslated region (UTR) bears an internal ribosomal entry site (IRES) that directs the landing of the ribosomes internally without scanning from the 5' end (8,9). A set of canonical and noncanonical (IRES-specific) translation factors is required for efficient IRES-mediated translation (10).

The requirement of specific proteins during internal initiation of translation varies in different viruses, and the identification of such proteins is an important step in understanding the molecular mechanism of the process. Furthermore, it has been proposed that the interaction with some of these factors determines tissue tropism and pathogenesis (11-13). Therefore, an initial and important question to be addressed is whether the oocyte translation machinery is capable of recognizing and translating the viral RNA of interest. As an example, we describe below a procedure to isolate poliovirus RNA from infected cells and to test the ability of oocytes to translate this viral RNA.

3.1.1. Purification of Poliovirus RNA From Virions

1. Infect a suspension culture of HeLa S3 cells (2 x 107 cells, about 50 mL of confluent culture) by adding 500 |L poliovirus stock (1 x 108 viruses).

2. Harvest cells 15 h postinfection and lyse the cells (50 mL) by three cycles of freezing and thawing in the same tissue culture medium.

3. Clarify the lysate by centrifugation at 9000g for 5 min and use the total volume to infect a suspension culture of 4 x 108 cells (1 L).

4. Incubate the infected cells at 37°C for 7 h and harvest them by centrifugation at 2000g for 10 min.

5. Wash the cell pellet with cold phosphate-buffered saline (PBS) and lyse the cells in 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, and 0.1% Nonidet P-40. Nuclei and cellular insoluble debris are removed by centrifugation at 10,000g for 10 min. SDS is added to the supernatant to a final concentration of 0.5%.

6. Precipitate viruses by centrifugation for 1 h at 200,000g. Precipitated viruses should be resuspended overnight in 2 mL TSE buffer.

7. Purify viruses by CsCl gradient. To prepare 10 mL gradient, mix 1 mL of nonionic detergent Brij 10%, CsCl to final density S = 1.33 g/cc in TSE-0.5% SDS, and 2 mL viral pellets. Spin the gradient 17 h at 35,000 to 40,000g at 20°C. Collect 500-|L fractions and measure optical density (OD) at 280 nm. The virus will be recovered in the middle of the gradient (S = 1.35 g/cc). Pool fractions containing the virus and dialyze against TSE buffer. Treat the sample with proteinase K (200 |g/mL) to release the RNA.

8. Extract viral RNA with phenol-chloroform (1/1). Add 2.5 volumes of ethanol, 1/10 volumes of 3M sodium acetate, incubate 5 min on dry ice, and spin at 14,000 rpm for 5 min in a microfuge. Resuspend the pellet in TE buffer and store at -70°C. It is recommended to confirm the integrity of the RNA by electrophoresis of an aliquot on 0.7 to 1% agarose gels and visualize by ethidium bromide.

Similar procedures can be use to purify RNA from other picornaviruses using the appropriate host cell.

3.1.2. Microinjection of Viral RNA Into Oocytes

Oocytes are surgically isolated and enzymatically defolliculated by incubation with 2 mg/mL collagenase for 3 h at room temperature. Defolliculated oocytes are washed five times with MBS and kept in MBS at 17 °C. Stage VI oocytes should be sorted for microinjection. Examine the oocytes under the dissecting microscope and separate good oocytes, which are large and have a symmetrical, even pigmentation. The animal hemisphere should be dark brown or black without white spots. The vegetal hemisphere should be yellow to pale white. To evaluate translation of the viral RNA, the oocytes can be labeled with [35S] methionine as follows:

1. Microinject about 100 oocytes with 25 nL viral RNA (0.5 |g/|L) each using a standard microinjection apparatus (we use the Nanoliter 2000, which delivers variable volumes from 5 to 60 nL).

2. For protein labeling, microinjected oocytes should be incubated in MBS containing 400 | Ci [35S] methionine per milliliter of media.

3. Take 20 oocytes at different times after microinjection (from 2 to 24 h) and lyse them "mechanically" in 200 |L buffer H.

4. Remove debris by centrifugation at 5000g for 5 min, and proteins can be immunoprecipi-tated using antibodies directed against viral proteins using standard procedures (see Note 1).

5. Immunoprecipitated complexes should be resuspended in Laemmli sample buffer, incubate at 100°C for 5 min, and analyze by SDS polyacrylamide gel electrophoresis (PAGE).

- HeLa cell proteins + HeLa ceit proteins Time (h) 10 20 30 10 20 30

Fig. 2. Translation of poliovirus RNA in Xenopus oocytes requires additional host factors. SDS-PAGE analysis of [35S]-labeled viral proteins synthesized in oocytes is shown. Oocytes were microinjected with poliovirus RNA in the absence (lanes 1-3) or presence (lanes 4-6) of HeLa cell extracts and incubated with [35S] methionine at 22°C for 10 h (lanes 1 and 4), 20 h (lanes 2 and 5), or 30 h (lanes 3 and 6). Cytoplasmic extracts were immunoprecipitated with antiserum directed against capsid proteins and analyzed on gels. The relative migration of poliovirus capsid proteins (P1, Vp0, Vp2, and Vp3) is indicated.

To analyze whether specific host factors are required for viral RNA translation in oocytes, cytoplasmic extracts obtained from specific cell lines can be coinjected with the viral RNA. Next, we describe the procedure to prepare cytoplasmic extracts from HeLa cells.

1. Harvest 4 x 107 cells (100 mL suspension culture) by centrifugation and wash three times with cold PBS.

2. Resuspend the cell pellet in 2 vol of hypotonic buffer, incubate on ice for 20 min, and then disrupt cells with 20 strokes with a glass Dounce homogenizer.

3. Obtain a postnuclei supernatant by centrifugation at 5000g for 10 min at 4°C. This supernatant is submitted to a second centrifugation (15,000g for 20 min). The resulting supernatant contains approx 10 |lg/|lL of cytoplasmic proteins.

4. Transfer the supernatant to a clean tube and store at -70°C.

To evaluate the effect of cytoplasmic extracts on viral translation efficiency, the viral RNA is mixed with different amounts of the cytoplasmic extract just before microinjection. In the case of HeLa cell extracts, it is possible to mix viral RNA with cytoplasmic proteins up to a ratio of 1/1 v/v; however, different cell lines may contain variable amounts of ribonucleases that could degrade the RNA during the microinjection process. In those cases, two microinjections into the same oocytes, one with the viral RNA and a second one with the cell cytoplasmic extract, is recommended. The PAGE analysis of poliovirus protein synthesized in microinjected oocytes in the presence and absence of cytoplasmic extracts is shown in Fig. 2.

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