Vaccination against Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Using a Baculovirus Recombinant Vaccine Provides Durable Immunity in Rabbits.

Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly once introduced in a rabbit population. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice.

Zika virus (ZIKV) is a mosquito-borne virus that has a high risk of inducing Guillain-Barré syndrome and microcephaly in newborns. Because vaccination is considered the most effective strategy against ZIKV infection, we designed a recombinant vaccine utilizing the baculovirus expression system with two strains of ZIKV envelope protein (MR766, Env_M; ZBRX6, Env_Z). Animals inoculated with Env_M and Env_Z produced ZIKV-specific antibodies and secreted effector cytokines such as interferon-γ, tumor necrosis factor-α, and interleukin-12. Moreover, the progeny of immunized females had detectable maternal antibodies that protected them against two ZIKV strains (MR766 and PRVABC59) and a Dengue virus strain. We propose that the baculovirus expression system ZIKV envelope protein recombinant provides a safe and effective vaccine strategy.

Baculovirus-derived influenza virus-like particle confers complete protection against lethal H7N9 avian influenza virus challenge in chickens and mice.

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV).

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1-660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens' immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394-608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394-608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461-544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

RNAi-based immunity in insects against baculoviruses and the strategies of baculoviruses involved in siRNA and miRNA pathways to weaken the defense.

Protection against viral infection in hosts concerns diverse cellular and molecular mechanisms, among which RNA interference (RNAi) response is a vital one. Small interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI interacting RNAs (piRNAs) are primary categories of small RNAs involved in RNAi response, playing significant roles in restraining viral invasion. However, during a long-term coevolution, viruses have gained the ability to evade, avoid, or suppress antiviral immunity to ensure efficient replication and transmission. Baculoviruses are enveloped, insect-pathogenic viruses with double-stranded circular DNA genomes, which encode suppressors of siRNA pathway and miRNAs targeting immune-related genes to mask the antiviral activity of their hosts. This review summarized recent findings for the RNAi-based antiviral immunity in insects as well as the strategies that baculoviruses exploit to break the shield of host siRNA pathway, and hijack cellular miRNAs or encode their own miRNAs that regulate both viral and cellular gene expression to create a favorable environment for viral infection.

PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen's kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

An Integrated Platform for Serological Detection and Vaccination of COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.

Characterization of the Gene Expression Patterns in the Murine Liver Following Intramuscular Administration of Baculovirus.

Intramuscular administration of wild-type baculovirus is able to both protect against Plasmodium sporozoite challenge and eliminate liver-stage parasites via a Toll-like receptor 9-independent pathway. To investigate its effector mechanism(s), the gene expression profile in the liver of baculovirus-administered mice was characterized by cDNA microarray analysis. The ingenuity pathway analysis gene ontology module revealed that the major gene subsets induced by baculovirus were immune-related signaling, such as interferon signaling. A total of 40 genes commonly upregulated in a Toll-like receptor 9-independent manner were included as possible candidates for parasite elimination. This gene subset consisted of NT5C3, LOC105246895, BTC, APOL9a/b, G3BP3, SLC6A6, USP25, TRIM14, and PSMB8 as the top 10 candidates according to the special unit. These findings provide new insight into effector molecules responsible for liver-stage parasite killing and, possibly, the development of a new baculovirus-mediated prophylactic and therapeutic biopharmaceutical for malaria.

Efficient application of a baculovirus-silkworm larvae expression system for obtaining porcine circovirus type 2 virus-like particles for a vaccine.

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.

Induction of adaptive immune responses against antigens incorporated within the capsid of simian virus 40.

Simian virus 40 (SV40) is a monkey polyomavirus. The capsid structure is icosahedral and comprises VP1 units that measure 45 nm in diameter. Five SV40 VP1 molecules form one pentamer subunit, and a single icosahedral subunit comprises 72 pentamers; a single SV40 VP1 capsid comprises 360 SV40 VP1 molecules. In a previous study, we showed that an influenza A virus matrix protein 1 (M1) CTL epitope inserted within SV40 virus-like particles (VLPs) induced cytotoxic T lymphocytes (CTLs) without the need for an adjuvant. Here, to address whether SV40 VLPs induce adaptive immune responses against VLP-incorporated antigens, we prepared SV40 VLPs containing M1 or chicken ovalbumin (OVA). This was done by fusing M1 or OVA with the carboxyl terminus of SV40 VP2 and co-expressing them with SV40 VP1 in insect cells using a baculovirus vector. Intraperitoneal (i.p.) or intranasal administration of SV40 VLPs incorporating M1 induced the production of CTLs specific for the M1 epitope without the requirement for adjuvant. The production of antibodies against SV40 VLPs was also induced by i.p. administration of SV40 VLPs in the absence of adjuvant. Finally, the administration of SV40 VLPs incorporating OVA induced anti-OVA antibodies in the absence of adjuvant; in addition, the level of antibody production was comparable with that after i.p. administration of OVA plus alum adjuvant. These results suggest that the SV40 capsid incorporating foreign antigens can be used as a vaccine platform to induce adaptive immune responses without the need for adjuvant.

Influenza vaccine efficacy induced by orally administered recombinant baculoviruses.

Although vaccine delivery through the oral route remains the most convenient and safest way for mass immunization purposes, this method is limited by the requirement for large antigen doses and low vaccine efficacy. In this study, we generated recombinant baculoviruses (rBVs) expressing influenza hemagglutinin (A/PR/8/34) and orally delivered a low dose of rBVs to evaluate its vaccine efficacy in mice. Intranasal rBV vaccination was included in the whole experiment for comparison. We found that oral vaccination elicited high levels of virus-specific IgG and IgA antibody responses in both serum and mucosal samples (lung, tracheal, intestinal, fecal and vaginal). Surprisingly, complete protection from the lethal influenza challenge was observed, as indicated by reductions in the virus titer, inflammatory cytokine production, body weight change, and enhanced survival. These results suggest that oral delivery of the influenza rBV vaccine induces mucosal and systemic immunity, which protect mice from the lethal influenza virus challenge. Oral delivery of baculovirus vaccines can be developed as an effective vaccination route.

Epitope mapping of African swine fever virus (ASFV) structural protein, p54.

In the absence of a vaccine for African swine fever virus (ASFV), diagnostic tools are critical for early detection and implementation of control measures. Along with other immunogenic proteins, p54 is a good serological target for conducting ASF detection and surveillance. In this study, a panel of 12 mouse monoclonal antibodies (mAbs) was prepared against a baculovirus-expressed p54(60-178) polypeptide. Further screening showed that five mAbs were positive for reactivity against ASFV-infected cells and recombinant p54 proteins. Mapping studies using five polypeptides and 12 oligopeptides, showed that mAb #154-1 recognized a conserved polypeptide sequence, p54(65-75), and was placed into Group 1. Mabs #143-1 and #7 recognized a region covered by p54(93-113) and were placed into Group 2. Group 3 consisted of mAbs #101 and #117, which recognized p54(118-127). Sera from pigs infected with the low virulent OURT 88/3 strain recognized the same p54 region covered by the Group 3 mAbs. When tested in a neutralization format, only mAb #143-1 showed neutralization activity above background. Together, the results identify important antigenic and immunogenic regions located on p54, which provide new tools for improving ASFV diagnostics.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system.

The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus.

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine. The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.

Emerging Technologies for Low-Cost, Rapid Vaccine Manufacture.

To stop the spread of future epidemics and meet infant vaccination demands in low- and middle-income countries, flexible, rapid and low-cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high-yield yeast recombinant protein vaccines; b) insect cell-baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale-up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.

Baculovirus as a Tool for Gene Delivery and Gene Therapy.

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors.

Molecular mechanism of poliovirus Sabin vaccine strain attenuation.

Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.