KL, TL, and VL conceived and designed the experiments. KL and VL performed the experiments. KL, VL, and JY analyzed the data. KL, TL, and VL contributed reagents/materials/analysis tools. KL, VL, and JY wrote the paper.
¤a Current address: Department of Chemical Engineering and The Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, California, United States of America
The authors have declared that no competing interests exist.
Live-virus vaccines activate both humoral and cell-mediated immunity, require only a single boosting, and generally provide longer immune protection than killed or subunit vaccines. However, growth of live-virus vaccines must be attenuated to minimize their potential pathogenic effects, and mechanisms of attenuation by conventional serial-transfer viral adaptation are not well-understood. New methods of attenuation based on rational engineering of viral genomes may offer a potentially greater control if one can link defined genetic modifications to changes in virus growth. To begin to establish such links between genotype and growth phenotype, we developed a computer model for the intracellular growth of vesicular stomatitis virus (VSV), a well-studied, nonsegmented, negative-stranded RNA virus. Our model incorporated established regulatory mechanisms of VSV while integrating key wild-type infection steps: hijacking of host resources, transcription, translation, and replication, followed by assembly and release of progeny VSV particles. Generalization of the wild-type model to allow for genome rearrangements matched the experimentally observed attenuation ranking for recombinant VSV strains that altered the genome position of their nucleocapsid gene. Finally, our simulations captured previously reported experimental results showing how altering the positions of other VSV genes has the potential to attenuate the VSV growth while overexpressing the immunogenic VSV surface glycoprotein. Such models will facilitate the engineering of new live-virus vaccines by linking genomic manipulations to controlled changes in virus gene-expression and growth.
The engineering of viral genomes provides ways not only to explore viral regulatory mechanisms at a genomic level, but also to produce recombinant viruses that may serve as vaccines, gene delivery vectors, and oncolytic (tumor-killing) agents. However, the complexity of interactions among viral and cellular components involved in the life cycle of a virus can make it challenging to anticipate how altering viral components will influence the overall behavior of the virus. Lim, Lang, Lam, and Yin have developed a computer model that begins to mechanistically account for key virus–cell interactions in its predictions of viral intracellular development. Lim et al.'s model was able to capture experimentally observed effects of gene rearrangements on the levels and timing of viral protein expression and virus progeny production, aspects that are important for the design of live-virus vaccines. Refinement and extension of their approach to current and other virus systems has the potential to advance the application of viruses as therapeutic agents.
Infections caused by viruses persistently threaten human health. For example, 40 million, 350 million, and 170 million people in the world are carrying human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV), respectively [
During the last decade the emergence of reverse genetics techniques has created unprecedented opportunities to better control viral attenuation [
How can a detailed model for the intracellular growth of a virus be used to explore the behavior of mutant viruses that encode alternative designs? As a starting point, one can create alternative designs that reorder or rearrange the wild-type genes or regulatory elements. Such genomic changes can alter the timing and level of expression of different viral genes, and thereby impact growth because the production of viral progeny depends on the dynamic expression of viral genes. Preliminary models of such alternative genome designs can use the “language” of the wild-type virus. They retain the parameters that characterize wild-type molecular interactions, wild-type average rates of viral polymerase elongation, and wild-type composition of progeny viruses, but they apply them in a manner that reflects the reordering of wild-type components in the engineered genome. For example, the timing of expression for the genes of phage T7 during infection maps closely to their sequential order on the T7 genome [
VSV is a prototype negative-sense single-stranded RNA (
Here we develop an in silico model of a VSV infection cycle, incorporating known regulatory interactions and mechanisms and relevant quantitative data from the literature of the past 40 years. These interactions and the corresponding equation formulations are described in detail in the model development section of
Using our model with the established parameter set (
Model Parameters
Protein Composition of VSV Particle and Lengths of Its Encoded Products
The partial transcription termination mechanism (or attenuation) is common in (-)ssRNA viruses. This mechanism is important to satisfy the different needs of each viral protein during its infection cycle. Five attenuation factors for each intergenic region of the VSV genome (
Owing to the step-wise release of polymerases from each gene junction, our simulations estimated the gradual decrease of VSV mRNA synthesis in the order of N > P > M > G > L (
All the viral proteins that are in the cytoplasm, plasma membrane, or assembled virion particles are counted.
(A) Viral mRNAs.
(B) Viral proteins.
(C) Estimated levels of free viral proteins in infected BHK cells. Free viral proteins include all the proteins in the cytoplasm or plasma membrane, but exclude the proteins that are incorporated into nucleocapsids and virion particles. Vex(0) = 3.
Anti-genome templates are only utilized to amplify genomes, while genome templates are used to amplify both anti-genomes and mRNAs, and they are also incorporated into virion progeny particles. The higher demands for genome by these multiple tasks are satisfied by the stronger promoter of the anti-genome template compared with that of the genome template [
Virion and (-)NC indicate viral genomes that are incorporated into virion particles and intracellular nucleocapsids, respectively. (+)NC indicates anti-genomes that are incorporated into intracellular nucleocapsids. Vex(0) = 3.
The virion production rate in BHK cells is at maximum 5–10 h post-infection. In infected DBT cells, similar simulation results were obtained except that the synthesis of genome-sized viral RNAs continued for longer time (active until 15 h post-infection,
Genomic nucleocapsids can either be used as templates for RNA synthesis or they may be incorporated into progeny virions. Their fate depends on levels of polymerase and M protein, which respectively favor RNA synthesis or virion production pathways, as well as on the extent to which association of the nucleocapsid with M protein will dominate over association with polymerase, described with the parameter
During the infection cycle, virus actively and passively competes with the host for limited translation resources by inhibiting host transcription and by amplifying viral mRNAs, respectively. Viral leader–mRNA and M protein play key roles in this inhibition [
(A) The estimated distributions between active (equipped with the required accessory factors) and inactive (not equipped) ribosomes, and between ribosomes available for viral and host mRNAs are shown.
“- Time window -” indicates the time period during which viral translation is actively supported by host ribosomes.
(B) The estimated numbers of ribosomes available for viral translation and actually occupied by viral mRNAs are shown for the cases of infected BHK and DBT cells. Non-occupied ribosomes are considered as free ribosomes. When two lines coincide, no free ribosomes exist (all the available ribosomes are occupied by viral mRNAs). Vex(0) = 3.
For vaccine applications, one seeks to minimize viral pathogenicity and maximize its immunogenicity. Based on observed correlations between in vitro and in vivo results, we assume here that the pathogenicity and the immunogenicity of a virus are directly linked to the levels of progeny production [
The stepwise decline in the transcription of genes more distant from the 3′-end region promoter highlights how gene order affects gene expression in VSV. Advances in reverse genetics have made it possible to create gene-rearranged virus strains where the transcriptional attenuation mechanism then creates altered levels of gene expression [
The expression rate of each protein was normalized by that of N protein. Therefore, the relative expression rate of N protein is defined as one. X and Y coordinates of datapoints indicate the results of experiments and simulations, respectively. All the experimental datapoints were obtained from the literature [
We also employed our model to predict the growth of VSV strains having the N gene at four different locations (N1 [wild-type], N2, N3, and N4) and then compared the simulation results with the experimental data. N1, N2, N3, and N4 VSV strains have the gene orders, 3′-N-P-M-G-L-5′ (N1), 3′-P-N-M-G-L-5′ (N2), 3′-P-M-N-G-L-5′ (N3), 3′-P-M-G-N-L-5′ (N4), respectively. As shown in
(A) Experimental data.
(B) Simulation results. The growth of the N1 VSV strain is the fitting result, but the growth of the other three strains is the model prediction result. Vex(0) = 3.
The genome and anti-genome of VSV are synthesized in unequal amounts, determined by the differing strengths of their promoters [
(A) The balance of the production between genome and anti-genome is determined by the promoter strength of the anti-genome relative to that of the genome (
(B) The extension of phenotypic variations by double genomic manipulations was predicted. As an important phenotype of virus for vaccine use, the changes of virion production by the relocation of N gene along with the variation in the promoter strength of the anti-genome relative to that of the genome (
We speculate that a rational way to attenuate the pathogenicity of the live wild-type virus would be to swap its two promoters, giving an
Several variant VSV strains, including N1 through N4, have been made by Ball and Wertz [
To elicit a systematic immune response, live viral vaccines must present or display neutralizing epitopes, typically through the expression of viral surface proteins. Higher levels of antigen expression have been found to correlate with more rapid and potent induction of anti-viral antibodies [
The intracellular level of G protein is highly dependent upon the location of G gene on the viral genome. In the late infection stage, a large fraction of G proteins are incorporated into progeny particles, which significantly decreases the intracellular protein level. Vex(0) = 3.
For vaccine use we might aim to maximize the immunogenicity of VSV or a VSV-based vector through the expression increase of VSV G gene or inserted foreign gene while minimizing their potential pathogenicity by growth attenuation. Given such design goals, specifically for a VSV vaccine, we might prefer strain GPM, which showed the highest expression of G protein (
Seeking a more detailed correlation between locations of the two genes and the viral phenotypes relevant to vaccine application, we simulated in silico the growth of all mutants that retain the gene order P – M – L of the wild-type, but allow G and N to move, criteria that define 20 possible gene-order permutations. The viral growth and the level of G protein in infected BHK cells mainly depend on the locations of N gene and G gene, respectively (
(A) Virion production.
(B) G protein expression.
The size of each circle shows the relative level for each VSV strain, and the coordinate of each circle indicates the gene order in each strain's genome. The gray and white circles denote the cases of wild-type (N1G4) and non–wild-type strains, respectively. Under the line there is no VSV strain case (e.g., N1G1, N2G2, etc.). Vex(0) = 3.
Burst Size of VSV Strains
The changes of protein expression levels by gene shuffling can be a rational means to modify the viral features for vaccine use. Robust synthesis of antigen by a highly attenuated strain appears to be an effective vaccine strategy as Flanagan et al. previously suggested. In addition to controlled attenuation of virus growth, a potent vaccine should ideally elicit a strong humoral or cell-mediated immune response.
In the era of highly advanced genetic technologies, we have witnessed a turning point for the development of live viral vaccines. Conventional empirical vaccine development processes are now being replaced by more rational reverse-genetics–based ones. With this trend, much attention will be focused on mechanism-based design of less pathogenic and more immunogenic virus stains. Mathematical models for intracellular virus growth can support this design process by providing a tool to systematically analyze the viral infection regulatory network, identify critical regulatory mechanisms or components for redirecting viral phenotypes, and reverse engineer desirable phenotypes.
BHK cells were obtained from I. Novella (Medical College of Ohio, Toledo, Ohio, United States) and grown as monolayers at 37 °C in a humidified atmosphere containing 5% CO2. BHK growth medium was Minimal Essential Medium with Earle's salts (MEM) (Cellgro, Fisher Scientific,
Cells were harvested, resuspended in growth medium, and plated into six-well plates at a concentration of 5 × 105 cells per 2 ml per well. Plated cells were returned to the incubator and allowed to grow overnight. The next day, two representative cell monolayers were harvested and counted to give an approximate number of cells per well. Each monolayer was then incubated with 200 μl of virus inoculum (MOI 3) for 1 h to allow virus adsorption. The plates were rocked gently every 20 min to evenly distribute virions on the monolayers during the adsorption step. After the adsorption period, the monolayers were rinsed twice with 1 ml of HBSS and then placed under 2 ml of infection medium for incubation. Medium samples of 200 μl including virion particles were taken from each well at 2, 3, 4, 6, 8, 10, and 20 h post-inoculation. Samples were kept frozen at −90 °C until their analysis by the plaque assay.
BHK cells were plated into six-well plates and cultured to 90% confluence. Culture medium was removed from each well and replaced with 200 μl of serially diluted viral samples. The inoculated monolayers were returned to the incubator for 1 h to allow virus adsorption. The plates were rocked gently every 20 min. At the end of the adsorption period, the inoculum was removed from each monolayer sample and then replaced with 2 ml of agar overlay. The agar overlay consisted of 0.6% weight/volume (w/v) agar (Agar Nobel, Difco, BD Diagnostic Systems,
Using algebraic and differential forms of equations, our mathematical model aims to account for established molecular processing steps in the development of VSV. Most model parameters were extracted from the literature. However, five parameters were obtained by fitting our simulation results to experimental data that were from the literature and our own experiments. Key model parameters are given in
As shown in
(A) Infection cycle of VSV.
(B) Segmentation of genome-size viral templates to simulate the spatial–temporal changes of polymerase concentration on the templates.
(C) VSV partial transcription termination (or attenuation) mechanism.
Following the release of the encapsidated genome and proteins into the cytoplasm, VSV transcription is initiated. The viral transcription was assumed to be independent of host–cell functions such as replication [
We simulate the transcription and replication processes by considering the spatial–temporal distribution of template-associated polymerases. We first partition the viral genome and anti-genome templates into 40 segments, excluding their 3′ and 5′ end regions, which are the leader (
Before estimating the polymerase flux, we need to figure out how the polymerase complex and M protein compete with each other for binding to the genomic nucleocapsids as well as how the polymerases bound to nucleocapsids are subsequently distributed to one of three possible tasks: transcription, replication of genome, or replication of anti-genome. In our model we assume that the genomic templates (negative-sense nucleocapsids) whose promoters (leader regions) are free of polymerases are available for association with free polymerase or M protein. We further assume that the associations of the free genomic templates by M proteins or polymerases take place instantaneously:
The viral polymerase on the leader region of the genome starts either transcription or replication. If there are sufficient N proteins, transcription is inhibited by the encapsidation of nascent positive-sense RNAs by N proteins; then replication dominates transcription [
Our formulation for transcription assumes that the synthesis of viral mRNAs is rate-controlled by the transcription initiation as well as the elongation of polymerase. Transcription initiation rate is parameterized by the spacing between neighboring polymerases in our model. At a given polymerase elongation rate, the larger polymerase spacing indicates the lower rate of transcription initiation. Transcription initiation modulates the input of polymerases to the leader region of the genome.
We consider that both translation initiation and polypeptide chain elongation contribute to the rate of viral protein synthesis. The translation initiation rate is parameterized by the ribosomal spacing. In our model we first calculated the number of ribosomes involved in viral translation by considering the maximum concentration of the ribosomes bound to viral mRNAs at a fixed ribosomal spacing:
The ribosomes involved in viral translation (
The synthesis rate of each viral protein depends on the elongation rate of the ribosome, linear density of ribosomes on its corresponding mRNA, and its first-order decay rate:
We also accounted for the consumption of free N proteins during the encapsidation of genome-length nascent RNAs and assumed that the degradation of nucleocapsids yielded intact N proteins:
We assumed that N protein regulates the switch of the role of polymerase between transcription and replication by encapsidating the newly synthesized RNAs [
The level of polymerases that scan through the whole genome (
The synthesis and decay of genomic nucleocapsids are described in the same way as for those of the anti-genomic nucleocapsids except that the polymerases on the anti-genomic templates are not released at intergenic regions:
In our model, non-encapsidated nascent genome and anti-genome fragments are released from polymerases and immediately degraded.
As polymerases leave the promoter regions by moving toward the downstream sequences, the concentration of polymerases on the promoters will decrease. The dynamic changes of the polymerase concentrations on the promoters of the genomic and the anti-genomic templates are finally described, respectively:
We assume that the condensation of negative-sense nucleocapsid by M protein initiates the virion assembly and the condensed nucleocapsids are not degraded in the same manner as virion progeny. Whenever the requirement for the stoichiometric amounts of proteins is satisfied, progeny virions are instantaneously assembled and released to the extracellular space. The time required for the condensation of the negative-sense nucleocapsid, the assembly, and the budding of progeny virion was assumed to be negligible relative to the preceding steps.
In our model, the host cell provides unlimited building blocks such as nucleoside triphosphates and amino acids for the growth of virus. However, as viral components accumulate during the course of infection, some key host components for translation such as initiation and elongation factors may be depleted [
The inhibition by the leader mRNA causes a first-order decay of the host factors, resulting in a shortage of the ribosomes equipped with the accessory factors for viral translation in the late infection stage in our model. Unlike viral transcription and replication, viral translation is directly affected by the decay of host factors since it depends entirely on host machinery. In the early infection, host mRNAs outnumber viral mRNAs and thereby successfully compete for the host translation machinery. However, the newly synthesized M proteins inhibit the host transcription initiation and the export of host mRNAs from the nucleus to the cytoplasm [
Considering the decay of host factors and the competition between host and viral mRNAs, we could derive a formula to quantify the number of the fully functional ribosomes that are available for the viral protein synthesis over time post-infection (
Although the ribosomes distribute into membrane-bound and cytoplasmic forms, each class supporting the syntheses of the viral G protein and the other four viral proteins (N, P, M, and L), respectively, we treated the ribosomes in our model as one population.
The initial condition for our simulation is set by a fixed number of infectious extracellular virus particles per cell (
Nomenclature
The relative strength of the anti-genomic promoter relative to the genomic promoter is given by
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Two host parameters (
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For each value of
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We thank Andrew Ball and Gail Wertz for helpful discussions and for providing recombinant virus strains.
¤b Current address: Institut für Mechanische Verfahrenstechnik, Universität Stuttgart, Stuttgart, Germany
¤c Current address: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America
amino acids
baby hamster kidney
chloramphenicol acetyl transferase
delayed brain tumor
envelope
polymerase
matrix
nucleocapsid
nucleotides
phospho
vesicular stomatitis virus
weight/volume