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Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells - PubMed

Comparative Study

Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells

Stephanie A Campbell et al. J Virol. 2005 May.

Abstract

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.

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Figures

FIG. 1.
FIG. 1.

Expression patterns of neuronal marker proteins in HeLa (human cervical carcinoma), HEK 293 (human embryonic kidney), Vero (African Green Monkey kidney), Sk-N-Mc (human neuroblastoma), Sh-SY5Y (human neuroblastoma), and DMS79 (human small cell lung cancer) cells. Cell extracts were prepared as described in Materials and Methods and utilized for five Western blot analyses of the light (NF-L), medium (NF-M), and heavy (NF-H) neurofilament subunits, neuron-specific enolase (NSE), and tau protein.

FIG. 2.
FIG. 2.

Genetic structure (A), one-step growth curves (B), and viral gene expression (C) of PV1/M(IRES), PV1/S(IRES), and RPS. (A) The heterologous HRV2 IRES is outlined by a gray box, and the asterisk indicates the approximate position of the attenuating mutation at nt 480 in SLD V of the PV1(S) IRES. Individual 5′ UTR SLDs are indicated by Roman numerals atop. (B) One-step growth kinetics in HeLa (▪), Sk-N-Mc (□), and HEK 293 (•) cells were established as described in Materials and Methods. (C) The accumulation of viral gene products was assessed by Western blot detection of the polioviral proteins 2C/2BC at various hours p.i.

FIG. 3.
FIG. 3.

Genetic structure (A), one-step growth curves (B), and viral gene expression (C) of PV1/M(δ6), PV1/S(δ6), and RPS(δ6). (A) The respective IRESes were manipulated by placing the conserved cryptic AUG codon (boxed black) in optimal Kozak context (…accAUGg…). This manipulation moved the translation initiation site upstream and deleted SLD VI. The sequence surrounding the artificial initiation codon of PV1/S(δ6) is identical to PV1/M(δ6). (B) One-step growth curves in HeLa (▪) and HEK 293 (•) cells. (C) Viral gene expression was assessed by Western blot detection of the polioviral proteins 2C and 2BC at various hours p.i.

FIG. 4.
FIG. 4.

Primary sequence and predicted secondary structure of the PV1(M), HRV2, and recombinant SLD Vs. Recombinant SLD Vs were inserted into the RPS(δ6) background, featuring HRV2 IRES SLD II-IV and deletion of SLD VI (see Materials and Methods). Nucleotides derived from PV sequence are shown in black boxes. (A) RPS(δ6-PV5) carries SLD V derived of PV1(M). Unboxed nucleotides are identical between HRV2 and PV1(M). The indicated positions of the attenuating mutations in the Sabin strains and the numbering refer to the sequence of PV1(M) (24). (B to D) RPS(δ6-PV5a-c) feature various sequence content derived of PV1(M) in the distal loop region of SLD V. (E) RPS(δ6) contains the HRV2 SLD V.

FIG. 5.
FIG. 5.

Genetic structure (A), viral propagation (B), and gene expression (C) of RPS derivatives carrying SLD Vs shown in Fig. 4. (A) The composition of SLD V for the respective constructs is schematically indicated. Refer to Fig. 4 for detailed sequence information. (B and C) Replication profile and time course of viral gene expression in HeLa (▪) and HEK 293 (•) cells.

FIG. 6.
FIG. 6.

Emergence of wt PV1(M) IRES sequence upon serial passage of PV1/S(IRES) in HEK 293 cells. The arrows indicate the position of nt 480 in SLD V of the PV1(S) IRES. The 5th passage contained mixed populations (G→A/G), while after 10 passages only the wt IRES (G→A) was detected. Sequence was generated using a 3′ primer and thus reads 3′ to 5′.

FIG. 7.
FIG. 7.

Translation of IRES reporter RNAs in HeLa, Sk-N-Mc, and HEK 293 cells. Capped RNA transcripts expressing fLuc under control of the β-globin leader (A) or uncapped IRES-controlled RNAs (B) were transfected into cells. (C) Translation of the capped control (black bars) and reporter constructs containing the HRV2 (grey bars) or PV1(M) IRES (white bars) was evaluated at several intervals posttransfection as described in Materials and Methods. The values are averages of triplicate samples, and the error bars indicate standard deviation.

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