key: cord-0002141-qe5lg8jn
authors: Lee, Cheri A.; August, Avery; Arnold, Jamie J.; Cameron, Craig E.
title: Polymerase Mechanism-Based Method of Viral Attenuation
date: 2015-06-01
journal: Vaccine Technologies for Veterinary Viral Diseases
DOI: 10.1007/978-1-4939-3008-1_6
sha: 7bbed13119c94625bd7b7b92a945ca65dc20afc7
doc_id: 2141
cord_uid: qe5lg8jn

Vaccines remain the most effective way of preventing infection and spread of infectious diseases. These prophylactics have been used for centuries but still to this day only three main design strategies exist: (1) live attenuated virus (LAV) vaccines, (2) killed or inactivated virus vaccines, (3) and subunit vaccines of the three, the most efficacious vaccines remain LAVs. LAVs replicate in relevant tissues, elicit strong cellular and humoral responses, and often confer lifelong immunity. While this vaccine strategy has produced the majority of successful vaccines in use today, there are also important safety concerns to consider with this approach. In the past, the development of LAVs has been empirical. Blind passage of viruses in various cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Also, due to the high error rate of RNA viruses and selective pressures of the host environment, these LAVs, derived from such viruses, can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs. One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase, which doesn’t lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation from a lysine to an arginine results in a high fidelity polymerase that replicates slowly thus creating an attenuated virus that is genetically stable and less likely to revert to a wild-type phenotype. This chapter provides detailed methods in which to identify the conserved lysine residue and evaluating fidelity and attenuation in cell culture (in vitro) and in the PV transgenic murine model (in vivo).

LAVs remain the most effective strategy for vaccine design [ 1 , 2 ] . However, in the past developments of these vaccines have been empirical. Blind passage of viruses in different cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Due to the high error rate of RNA viruses and the selective pressures of the host environment, these LAVs can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. LAV vaccines have been created against a number of RNA viruses, such as poliomyelitis, measles, mumps, rabies, rubella, yellow fever and infl uenza. While these vaccines have been successful there still remains a need for a rational design strategy in which to create additional LAVs. It has been shown that by altering fi delity, the rate and speed at which the polymerase incorporates mutations, leads to viral attenuation [ 3 -8 ] .

RNA viruses are defi ned by high mutation rates, high yields, and short replication times. These viruses have an average mutation rate of 10 −3 to 10 −5 mutations per genome replication event [ 4 ] . As a result, RNA viruses do not replicate as a single sequence but as a "cloud" of mutant genomes, which have been dubbed quasispecies [ 9 -12 ] . Although a high mutation rate can lead to deleterious changes in the genome, genetic diversity in RNA virus populations appears to be critical for fi tness and survival and likely contributes to pathogenesis. In a heterogeneous pathogen population, some variants are able to infect primary tissues and bypass host-imposed bottlenecks. From here the remaining variants can replicate into another heterogeneous population where some are once again able to bypass another layer of bottlenecks and perform secondary infection in other tissues thus demonstrating that a heterogeneous population, or quasispecies, can be benefi cial to the pathogen. This adaptability poses a unique challenge, for example, when it comes to developing antiviral drugs and vaccines.

RNA virus populations are heterogeneous due to error-prone replication by the viral RNA-dependent RNA polymerase (RdRp) which infl uences quasispecies evolution. This adaptability benefi ts the pathogen sometimes at the cost of the host. Currently, error-prone replication is known to happen in all RNA viruses that infect both plants and animals. It is also known that this error is due to rapid generation of variants and the fi delity of the viral RdRp [ 13 -15 ] .

In order to study the effect of polymerase mutants on RNA virus heterogeneity, we turn to a model RNA virus, poliovirus. Poliovirus (PV) belongs to the family Picornaviridae . This family consists of non-enveloped, positive single strand genomes many of which are important human and animal pathogens. The PV genome can be divided into three parts, the 5′-untranslated region (5′-UTR), a single open reading frame (ORF), and the polyadenylated 3′-untranslated region (3′ UTR). Upon entry into the cell, the mRNA is translated as a polyprotein of approximately 3000 amino acids and can be divided into three functionally different regions: P1, P2, and P3. The polyprotein is cleaved cotranslationally and posttranslationally by viral proteases 2A pro and 3C pro into 11 proteins. The PV RdRP is found in the P3 region and is termed 3Dpol.

The RdRp is one out of four categories of polymerases and its crystal structure shows a close evolutionary relationship not only to other RdRps but also to that of DNA-dependent DNA-polymerases (DdDps), DNA-dependent RNA-polymerases (DdRps), and RNAdependent DNA-polymerases (RdDps) also known as reverse transcriptases (RTs). All resemble a cupped right-handed structure consisting of the thumb, fi ngers, and palm subdomains [ 16 , 17 ] . The palm is where the active site of the polymerase lies and consists of four conserved structural motifs A-D [ 16 ] . A fi fth and sixth motif, E and F, exists in the RNA-dependent polymerases but not the DNA-dependent polymerases [ 16 ] . The latter motifs are not in the active site but line this region. RdRps are error prone but they are as faithful as DNA polymerases that lack proofreading exonucleases [ 18 ] . The absence of a repair mechanism in the PV genome is what leads to an enhanced rate of mutation during viral replication.

Nucleic acid polymerases use a two-metal-ion mechanism for nucleotidyl transfer [ 19 ] . In this mechanism, two magnesium ions are used to organize the reactants. Recently, the chemical mechanism of nucleotidyl transfer has been expanded to include a general acid, which protonates the pyrophosphate leaving group of the NTP substrate and enhance the effi ciency of nucleotidyl transfer [ 20 , 21 ] . The general acid of PV RdRp is Lys359, located in motif D, which is conserved throughout all RdRps and RTs. Importantly, an orthologous residue at this site is known or predicted in RNA viruses for which rational design of vaccines would greatly benefi t.

In order to determine what effect biochemical changes have on the multiplication of the virus in cell culture we created a PV genome encoding the arginine mutation in the PV subgenomic replicon (pRLucRA) and viral cDNA (pMoVRA). Quantifi cation of virus by plaque assay provides insight into fi tness of the viral population. The subgenomic replicon permits indirect evaluation of RNA synthesis by measurement of luciferase activity. Analysis of RNA replication in the absence of virus production can provide insight on whether RNA replication is the rate-limiting step for virus production.

The characteristics of live-virus multiplication and their plaque phenotype can predict whether the virus will be attenuated in the mouse model. However, viral quantifi cation by plaque forming units (pfu) selects variants based on phenotype and therefore can be an unreliable measure of viruses present due to phenotypic differences between viral strains. In addition to pfu, quantifying virus based on genomes accounts for total viral particles produced. This is a more accurate measurement of total viruses in the population and the number of genomes produced by the polymerase.

While these characteristics predict attenuation, actual confi rmation is determined using a mouse transgenic for the PV receptor. In this system, wild-type (WT) PV is generally lethal. At the highest dose, the mutated polymerase (lysine to arginine) failed to

cause disease in the mice. To determine if the mutant virus replicated, mice surviving the initial infection were challenged with a lethal dose of WT PV. We can conclude from the mice that survive this challenge, the mutant is replication competent and elicits an immune response suffi cient enough to protect against a lethal dose of WT PV [ 3 ] .

Using PV as our model we have developed a rational design for polymerase-based mechanism of attenuation. By altering the nature of the general acid lysine residue to an arginine, we have shown that we maintain the ability to tune RdRp speed and fi delity creating a viral RdRp that is slower and more faithful than the WT enzyme. This results in an attenuated virus with a restricted viral quasispecies that fails to cause disease, yet elicits a protective immune response. This approach has the ability to be applied to any RNA virus given the conserved nature of the motif D lysine residue. Table 1 is an alignment of residues found in motif D of the RdRp for positive and negative strand RNA virus families. Numbers indicate the position from the fi rst amino acid of motif D in the RdRp domain. The conserved lysine residue is shown in boldfaced type. Other conserved residues are underlined. All sequences were obtained from the NCBI Database. Sequences were aligned using ClustalW2 and based upon alignments previously published [ 22 ] . 

The polymerase gene was amplifi ed using pMoV-3D-BPKN plasmid as a template. This template has silent mutations engineered into the 3Dpol coding sequence. The "naked" viral cDNA, pMoVRA, contains 4 Pst I restriction sites and pRLucRA contains 3. A Pst I site was engineered into the 3Dpol coding sequence such that when cloned into the "naked" vectors, pMoVRA and pRLu-cRA, and digested with Pst I, positive clones containing the mutated PCR product will have 5 and 4 bands respectively when run on a agarose gel. Clones positive by restriction digest are verifi ed for the presence of the mutation by sequencing.

(a) External forward primer: PV-3D-BglII-for.

(b) Internal reverse primer: PV-3D-K359R-rev.

2. Perform amplifi cation reactions in three separate 100 μL volumes, fi nal concentration containing: 1× Thermopol buffer, 3 mM dNTPs, 0.5 μM of each primer, 0.5 ng/μL of template plasmid pMo-3D-BPKN, and 2 U of Deep Vent Polymerase (Table 2 ).

3. Cycling conditions consist of a preliminary denaturing step at 95 °C for 4 min followed by a hot start cycle for 4 cycles at 95, 50, and 72 °C each for 1 min and fi nally, 18-20 cycles of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min and product extension at 72 °C for 2 min and a fi nal product extension at 72 °C for 10 min.

4. Prepare 2, 1.2 % agarose gels.

5. The rest of product tubes are combined and DNA is precipitated with 100 % ethanol. Add 1/5th volume (60 μL) 3 M NaOAc, mix well with pipet then add three volumes (1080 μL) 100 % EtOH and mix well. Freeze mixture on dry ice until liquid is a slow moving "sludge" when inverted. Centrifuge at top speed for 10 min. You will observe a thick white pellet. (a) External reverse primer: PV-3D-EcoRI-ApaI-polyA-rev.

(b) Internal forward primer: PV-3D-K359R-for. 9 . Same procedure as for PCR reaction A (Table 3 ) . 10 . Repeat concentration and purifi cation a for PCR A.

1. Set up 3-100 μL reactions as before this time using PCR reactions A and B as templates (Table 4 ):

(a) External forward primer: PV-3D-BglII-for.

(b) External reverse primer: PV-3D-EcoRI-ApaI-polyA-rev.

2. Concentrate and purify PCR product as previously described for PCR products A and B. 

Bgl II. Digest purifi ed cDNA by adding 2 μg of DNA to a 1.5 mL tube containing 10 μL of the appropriate 10× restriction buffer with 4 μL (40 U) of enzyme in a total volume of 100 μL and incubate according to manufacturer's instructions. We recommend 2-4 h for an incubation time. For the purifi ed overlap PCR product, repeat the same procedure as above, using the entire 50 μL product in the digest (Table 5 ).

3. Allow reaction to proceed at 37 °C for 2 h.

4. Run a sample of uncut and cut plasmids on 1 % agarose gel to check effi ciency of reaction ( see Note 3 ).

5. When you have verifi ed that the plasmid has been linearized, purify with QIAEX II gel extraction kit. Follow the kit's protocol for purifying and concentrating DNA from an aqueous solution.

6. Clean up DNA using QIAEX II gel extraction kit. Follow the kit's protocol for purifying and concentrating DNA from an aqueous solution.

7. Suspend silica bead pellet in 50 μL T 10 E 0.1 and incubate at 65 °C for 10 min.

8. Quick spin the tube and remove both supernatant and beads and add to Spin-X fi lter tube. Spin at 800 × g for 5 min and collect eluted DNA from tube. Table 3 Round 1-2: extension PCR-reaction B

10× Thermopol reaction buffer 9. Digest purifi ed cut cDNA by adding entire 50 μL of cut DNA to a 1.5 mL tube containing 10 μL of the appropriate 10× restriction buffer with 1 μL (50 U) of enzyme in a total volume of 100 μL and incubate according to manufacturer's instructions. We recommend 2-4 h for incubation time. For the purifi ed cut overlap PCR product, repeat the same procedure as above ( 1. pMoV-3D-K359R and pRLuc-3D-K359R plasmids are fi rst linearized with restriction enzyme Apa I.

2. Digest purifi ed cDNA by adding 5 μg of mutant plasmid to a 1.5 mL tube containing 10 μL of the appropriate 10× restriction buffer with 2.5 μL (50 U) of enzyme in a total volume of 100 μL and incubate according to manufacturer's instructions. We recommend 2-4 h for incubation time (Table 8 ) .

3. Run a sample of uncut and cut plasmids on 1 % agarose gel to check effi ciency of reaction ( see Note 3 ).

4. When you have verifi ed that the plasmid has been linearized, purify with QIAEX II gel extraction kit. Follow the kit's protocol for purifying and concentrating DNA from an aqueous solution.

5. Suspend silica bead pellet in 50 μL T 10 E 0.1 and incubate at 65 °C for 10 min.

6. Quick spin the tube and remove both supernatant and beads and add to Spin-X tube. Spin at 800 × g for 5 min and collect eluted DNA from tube.

7. Add H 2 O fi rst; subtract the DNA volume from 2.5 μL to get the volume of H 2 O to be added.

8. Next add the following in this order to a total volume of 20 μL, fi nal concentration containing: 350 mM HEPES, 32 mM MgAcetate, 40 mM DTT, 2 mM spermidine, 28 mM NTPs, 0.5 μg linearized cDNA, and 0.5 μg T7 RNAP into an autoclaved 0.6 mL microcentrifuge tube (Table 9 ).

9. Pre-incubate the reaction mix at 37 °C for 5 min prior to adding T7 RNAP.

10. Add T7 RNAP.

11. Incubate reaction at 37 °C. After 30 min, check reaction for cloudy, white precipitate, which is magnesium pyrophosphate forming, to ensure the reaction is progressing. Allow reaction 

to incubate for 4-5 h, spin the reaction for 2 min to pellet out the magnesium pyrophosphate.

12. Transfer supernatant to a new tube and then add 2 μL of RQ1 DNase (2 U) and incubate for 30 min at 37 °C.

13. Clean up RNA using Qiagen RNeasy Mini Kit following manufacturer's instructions for RNA cleanup.

14. Measure concentration of purifi ed RNA product using a NanoDrop spectrophotometer. Store RNA at −80 °C until ready to use. 5. Add 400 μL of HeLa cell suspension to 1.7 mL tube of RNA. Quickly add the mixture to the cuvette and place cuvette into chamber and zap cells. Using media from 15 mL conical (pre-warmed from water bath) add 600 μL of media to zapped cells still in the cuvette. Gently pipet up and down multiple times to mix cells and media and to break up any cell clumps that may have formed. 3. Add 600 μL of media to zapped cells still in the cuvette. Gently pipet up and down multiple times to mix cells and media and to break up any cell clumps that may have formed.

4. Add cell and media mixture back to 15 mL conical tube. Close the tube and gently invert back and forth to mix. Aliquot 500 μL into a 1.7 mL tube and place the 15 mL conical in the 37 °C incubator until ready to take next timepoint.

5. Spin the one 1.7 mL tube at 2500 × g for 2 min for 0 h time point.

6. Remove media and wash pellet with 1× PBS. Spin again. Remove 1× PBS and add 100 μL of 1× CCLR (diluted to 1× in ddH 2 O). Vortex tube for 10 s and place on ice.

7. Repeat this for every time point taken and leave all cells on ice until the next day. Make that each time an 500 μL aliquot is removed from the 15 mL conical tube that the tube is inverted multiple times to ensure an even distribution of cells. 21. Incubated at 37 °C and harvest cells and media using a sterile disposable spatula (to scrape cells off the bottom of the well) at various time points post-infection. Virus and cell mixture is put into autoclaved 1.7 mL microcentrifuge tubes and immediately frozen on dry ice.

Harvest virus by three repeated freeze-thaw cycles and quantitate virus titers as described above.

23. Also extract RNA using Qiagen RNeasy Plus Mini Kit to determine genomes/mL by RT-qPCR.

3. Combine water, cDNA and buffer in 1.7 mL tube and before adding enzyme to your reaction, remove 5 μL from the tube and set aside. This will be your "uncut" sample. Add the enzyme and allow reaction to proceed at proper temperature. After incubation remove another 5 μL from the tube. This will be your "cut" sample.

4. The number of colonies after successful ligation and transformation of cloned pMoVRA and pRLucRA yield different results. When plating 100 % of your transformed cells, the pMoVRA clone should yield roughly 50 colonies on a plate, whereas the pRLucRA clone will yield at most 100 colonies.

5. Multiple freeze thaws of virus stock will overtime lower the titer of the virus. To avoid this, make small aliquots of virus and store at −80 °C. Never use a stock tube that has been thawed more than three times after a titer or genome copy has been determined.

6. There are two reasons why it is important to passage virus at a low MOI. First is to check the stability of the engineered mutation by sequencing the mutated region; second is to generate a quasispecies.

70 % ethanol solution: 70 % EtOH, 30 % ultrapure water

Omnipur Agarose (Millipore/Calbiochem)

5× TBE electrophoresis running buffer: 33 mM Tris-HCl, 40 mM boric acid, 1 mM EDTA

Using P0 virus, extract viral RNA using the QIAamp Viral RNA mini kit, following manufacturer's directions

Determine viral genome copies by performing RT-qPCR on extracted virus sample

Create a standard curve using in vitro transcribed RNA. Dilute RNA to 4 ng/μL, which is approximately 1 × 10 9 genome copies/μL. For a more accurate determination of genome copies/ μL, use digital PCR

HeLa S3 cells per well and cover with 3 mL complete media

Incubate virus and cells at 37 °C for 30 min. Remove virus and wash cells with 1 mL 1× PBS. Remove PBS and add 1 mL complete media to wells and allow viral replication to proceed for 8 h

Fecundity Assay Polymerase Mechanism-Based Method of Viral Attenuation

After 8 h, purify total RNA from infected cells with Qiagen RNeasy Plus Mini Kit

Harvest virus by three repeated freeze-thaw cycles. Perform RT-qPCR purifi ed RNA to calculate the amount of virus required to infect next HeLa cells

House 4-6 week old outbred (ICR) mice transgenic for the PV receptor (cPVR) in standard ventilated caging for all experiments

Passage 4 (P4) viral stocks are used for animal inoculations ( see Virus isolation, titer and one-step growth curves)

Generate all virus stocks in serum-free media, harvested, titered, and genomes obtained

Inoculate mice via intraperitoneal route (i

PD 50 s are performed by infecting fi ve mice per viral dose (1 × 10 7 , 1 × 10 8 , and 1 × 10 9 pfu) in order to calculate a PD 50

Rationalizing the development of live attenuated virus vaccines

Live attenuated infl uenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal infl uenza viruses during infection of human nasal epithelial cell cultures

A polymerase mechanism-based strategy for viral attenuation and vaccine development

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population

Coxsackievirus B3 mutator strains are attenuated in vivo

Engineering attenuated virus vaccines by controlling replication fi delity

RNA virus population diversity: an optimum for maximal fi tness and virulence

Motif D of viral RNA-dependent RNA polymerases determines effi ciency and fi delity of nucleotide addition

Selforganization of matter and the evolution of biological macromolecules

RNA virus mutations and fi tness for survival

Molecular Quasi-species

Quasispecies structure and persistence of RNA viruses

Host alternation of chikungunya virus increases fi tness while restricting population diversity and adaptability to novel selective pressures

Characterization of the elongation complex of dengue virus RNA polymerase: assembly, Polymerase Mechanism-Based Method of Viral Attenuation kinetics of nucleotide incorporation, and fi delity

Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds

Structure of the RNA-dependent RNA polymerase of poliovirus

Structure-function relationships among RNAdependent RNA polymerases

From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity

Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes

Nucleic acid polymerases use a general acid for nucleotidyl transfer

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNAand DNA-dependent RNA and DNA polymerases

Identifi cation of four conserved motifs among the RNA-dependent polymerase encoding elements

Poliovirus RNA replication requires genome circularization through a protein-protein bridge

Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: age-dependent paralysis and a mucosal route of infection