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Journal of Virology, December 2007, p. 13552-13565, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01576-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of Venezuelan Equine Encephalitis Virus Capsid Protein Function in the Inhibition of Cellular Transcription

Natalia Garmashova,¹ Svetlana Atasheva,¹ Wenli Kang,² Scott C. Weaver,² Elena Frolova,^1,3 and Ilya Frolov^1*

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019,¹ Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609,² Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1072³

Received 19 July 2007/ Accepted 22 September 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The encephalitogenic New World alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses, constitute a continuing public health threat in the United States. They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that these viruses have developed the ability to interfere with cellular transcription and use it as a means of downregulating a cellular antiviral response. The results of the present study suggest that the N-terminal, 35-amino-acid-long peptide of VEEV and EEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of a cytopathic effect. The identified VEEV-specific peptide C_VEE33-68 includes two domains with distinct functions: the -helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and nucleus, and the downstream peptide, which might contain a functional nuclear localization signal(s). The integrity of both domains not only determines the intracellular distribution of the VEEV capsid but is also essential for direct capsid protein functioning in the inhibition of transcription. Our results suggest that the VEEV capsid protein interacts with the nuclear pore complex, and this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, cell death. The replacement of the N-terminal fragment of the VEEV capsid by its Sindbis virus-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but reduces cytopathogenicity and results in attenuation in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.

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The genus Alphavirus in the family Togaviridae includes a number of important human and animal pathogens (15). Alphaviruses are currently classified into six antigenic complexes and are widely distributed in both the New World and the Old World. They are efficiently transmitted by mosquitoes, in which they cause a persistent, lifelong infection with a minimal effect on biological functions. In vertebrates, alphavirus infection is acute and in many cases characterized by high-titer viremia, rash, fever, and encephalitis until the death of the infected host or clearance of the virus by the immune system. The encephalitogenic alphaviruses, including Venezuelan (VEEV), eastern (EEEV), and western (WEEV) equine encephalitis viruses, represent a continuous public health threat in the United States (41, 48-50). They circulate in Central, South, and North America and have the ability to cause fatal disease in humans and in horses and other domestic animals. During VEEV epizootics, equine mortality can reach 83%, and in humans, the virus produces a severe temporary immunodeficiency and a greatly debilitating, sometimes fatal disease (42). The overall mortality rate is below 1%, but neurological disease, including disorientation, ataxia, mental depression, and convulsions, can be detected in up to 14% of all infected individuals, especially children (23). Also described are sequelae of VEEV-related clinical encephalitis in humans (10, 27).

The VEEV genome is represented by a single-stranded RNA molecule of positive polarity that is almost 12 kb in length. It mimics the structure of cellular mRNAs, with a cap at the 5' terminus and a poly(A) tail at the 3' end of the RNA. The VEEV genome has been cloned in a cDNA form (24) that allows a wide variety of genetic manipulations to be undertaken.

The only experimental vaccine against VEEV infection that has been used extensively in humans was developed 4 decades ago by serial passaging of the virulent subtype IAB Trinidad Donkey VEEV strain in guinea pig heart cell cultures (3). Over 8,000 humans have been vaccinated during the past 4 decades (2, 6, 37), and the cumulative data indicate that nearly 40% of vaccinated people develop a disease with some symptoms typical of those seen with natural VEEV infection, including a febrile systemic illness and other adverse effects (2, 3, 21). No effective antivirals have been developed against any alphavirus, including VEEV.

In spite of the continuous threat of VEEV epidemics, the biology of this virus has been studied less intensively than those of other, less pathogenic alphaviruses, such as Sindbis (SINV) and Semliki Forest (SFV) viruses. These viruses can be readily manipulated in a low-biocontainment environment and represent good models for studying the mechanisms of alphavirus replication, virus-host interactions, and encephalitis development (14). However, important differences in pathogenesis and the severity of human and veterinary diseases suggest that these viruses may not be ideal models for encephalitis. Moreover, the results from recent studies of the Old World (SINV and SFV) and the New World (VEEV and EEEV) alphaviruses (1, 9, 11-13, 36, 46) demonstrated that both of these groups have developed the ability to interfere with cellular transcription and to use this effect as a means of downregulating cellular antiviral response. However, the mechanisms of transcription inhibition appear to be fundamentally different; while the Old World alphaviruses use nsP2 to inhibit cellular transcription (11), the more encephalitogenic VEEV and EEEV use their capsid proteins for the same function (1, 12). Expression of the latter proteins by different vectors is sufficient for induction of cell death and cytopathic effects (CPE) in tissue culture, and the development of these phenomena strongly correlates with the inhibition of transcription of cellular mRNA and rRNA. Moreover, the replacement of the structural protein genes in the VEEV genome by those derived from SINV has made the chimeric virus significantly less cytopathic and incapable of interfering with the development of an antiviral response in cells having no defect in alpha/beta interferon induction and signaling (12).

In this study, we continued our investigation of the VEEV capsid protein-dependent inhibition of cellular transcription. Our data demonstrate that (i) transcriptional shutoff and CPE development in capsid protein-expressing cells are determined by a short C_VEE peptide, C_VEE33-68, located in the N-terminal part of the protein; (ii) inhibition of transcription and CPE development correlate with the presence of the entire capsid protein or the indicated peptide on the nuclear membrane, which suggests their interaction with the nuclear pore complex (NPC); (iii) the mutations in the peptide might lead to the accumulation of capsid protein in either the cytoplasm or the nucleus; and (iv) VEEV variants encoding either the chimeric capsid protein with the amino-terminal fragment (amino acids [aa] 1 to 110) replaced by the corresponding SINV capsid-coding fragment or the VEEV capsid protein with the frameshift mutations changing a short peptide in the N terminus are highly attenuated but capable of very efficient replication in vitro in cells having defects in alpha/beta interferon signaling. This study provides new data at the cellular level on the mechanism of VEEV cytopathogenicity. These findings can be used for development of new, attenuated VEEV variants and may be applicable to other New World encephalitogenic alphaviruses.

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Cell cultures. BHK-21 cells were kindly provided by Sondra Schlesinger (Washington University, St. Louis, MO) and were maintained at 37°C in alpha minimum essential medium supplemented with 10% fetal bovine serum (FBS) and vitamins.

Plasmid constructs. The VEErepL replicon with a mutation in the nsP2 gene, Q₇₃₉L, was described elsewhere (12, 36). The distinguishing feature of this replicon is its noncytopathic phenotype and low levels of genome RNA replication and transcription of subgenomic RNA. Such replicons do not overproduce the proteins of interest and thus generate biologically relevant data. The VEErepL/GFP/Pac replicon, used as a noncytopathic control in many experiments, has been described elsewhere (11). The genes of the proteins of interest were cloned into VEErepL under the control of the subgenomic promoter, and a second promoter was introduced to drive the expression of puromycin acetyl transferase (Pac), which renders the replicon-containing cells resistant to translational arrest caused by puromycin. All of the tested cassettes expressing capsid proteins with different deletions were synthesized by PCR and sequenced before they were cloned into the vector replicon as green fluorescent protein (GFP) fusions. The selection of frameshift mutants of C_VEE has been described elsewhere (12), and the corresponding gene was synthesized by reverse transcription-PCR using RNA isolated from a replicon-containing cell line. In the GFP fusion proteins, the capsid-specific peptides were separated from the GFP by the short, flexible (Gly)₄ peptide. All of the GFP fusions were designed to avoid cleavage by the capsid-encoded protease activity. To achieve this, the last amino acid in the capsid protein was deleted. An additional AUG codon was added to all of the cassettes encoding VEEV- and EEEV-specific peptides that did not contain the initiating AUG. The VEEV/C_SIN1 plasmid encoded the infectious VEEV TC-83 genome, in which the sequence encoding aa 1 to 110 of C_VEE was replaced by the sequence encoding aa 1 to 98 of C_SIN. The VEEV/C_VEEfrsh genome encoded a capsid protein with previously identified frameshift mutations (12) affecting aa 58 to 85. All of the constructs that we used are presented in the corresponding figures. The sequences of the recombinant plasmids can be provided upon request.

RNA transcriptions. Plasmids were purified by centrifugation in CsCl gradients. Before being subjected to a transcription reaction, plasmids were linearized using the MluI or NotI restriction sites located downstream of the poly(A) sequence of the VEEV replicons. RNAs were synthesized by using SP6 RNA polymerase in the presence of a Cap analog as previously described (39). The yields and integrity of transcripts were analyzed by gel electrophoresis under nondenaturing conditions. RNA concentrations were measured on a FluorChem imager (Alpha Innotech), and transcription reactions were used for electroporation without additional purification.

Analysis of the cytotoxicities of the constructs. BHK-21 cells were electroporated by using previously described conditions (29). In all of the experiments, 5 µg of the in vitro-synthesized RNAs was used per electroporation of 5 x 10⁶ cells. Next, the aliquots of the cells were seeded into six-well Costar plates for analysis of cell proliferation and viability, as described elsewhere (11, 12). Puromycin selection (10 µg/ml) was performed between 6 and 48 h posttransfection. Then, the cells were incubated in puromycin-free media, and viable cells were stained with trypan blue and counted at the times indicated in the figures. In parallel, different dilutions of the electroporated cells were seeded into 100-mm tissue culture dishes. At 6 h posttransfection, puromycin was added to the media to a concentration of 10 µg/ml. Colonies of Pur^r cells were stained with crystal violet on days 4 to 9 posttransfection, depending on their growth rates. The results are presented in the figures in CFU per µg of RNA used for transfection.

Analysis of cellular transcription. BHK-21 cells were electroporated with 5 µg of the in vitro-synthesized RNAs, and 1/10 of the cells were seeded into 35-mm culture dishes. At 6 h posttransfection, puromycin was added to the media to a concentration of 10 µg/ml. At the indicated times postelectroporation, the cellular RNAs were labeled for the time periods given in the figure legends in complete alpha minimum essential medium supplemented with 10% FBS and 20 µCi/ml [³H]uridine. The RNAs were isolated with TRizol reagent as recommended by the manufacturer (Invitrogen) and analyzed by agarose gel electrophoresis as previously described (5). This assay does not allow one to discriminate between changes in RNA synthesis and RNA degradation; therefore, to additionally assess the cellular RNA synthesis, the RNA samples on Whatman 3MM paper were washed with cold 10% trichloroacetic acid. The radioactivity was measured by liquid scintillation counting and normalized for the number of viable cells, determined by the tests described above. Because we used the replicons with a mutated nsP2 gene, the replicon-specific RNA synthesis was less than 5% of the total RNA synthesis (data not shown). Replicon-specific RNAs could be detected in the gels only after very long exposures.

In all of the experiments, small fractions of the transfected cells were used to evaluate the levels of GFP fusion protein expression on Western blotting, using GFP-specific antibodies. No significant differences in expression were found; therefore, these data are not shown in the Results.

Infectious-center assay. Five micrograms of in vitro-synthesized, full-length RNA transcripts of viral genomes were used per electroporation. Tenfold dilutions of electroporated BHK-21 cells were seeded in six-well Costar plates containing subconfluent naïve cells. After 1 h of incubation at 37°C in a 5% CO₂ incubator, the cells were overlaid with 2 ml of minimum essential medium containing 0.5% Ultra-Pure agarose (Invitrogen) supplemented with 3% FBS. Plaques were stained with crystal violet after 2 days of incubation at 37°C.

Viral-replication analysis. To exclude any effect of possible virus evolution on replication efficiency, virus growth rates were evaluated directly after electroporation of the in vitro-synthesized RNA into the cells. BHK-21 cells were electroporated with 5 µg of the RNAs, and one-fifth of the cells were seeded into 35-mm culture dishes. At the indicated times posttransfection, the media were replaced by fresh media, and virus titers in the culture fluids were determined by a plaque assay on BHK-21 cells, as previously described (26).

Immunization and challenge with virulent VEEV. Female, 6-day-old NIH Swiss mice were inoculated intracerebrally (i.c.) with VEEV strain TC-83 or the designed mutants at a dose of 10⁶ to 10⁷ PFU in a total volume of 20 µl of phosphate-buffered saline. After infection, each cohort of 8 to 10 animals was maintained for 2 months without any manipulation. For 21 days, the mice were observed twice daily for signs of illness (ruffled fur, depression, anorexia, and/or paralysis) and/or death.

Microscopy. BHK-21 cells were seeded on glass chamber slides (Nunc) and transfected by 2 µg of in vitro-synthesized replicon RNA using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Then, at 12 h posttransfection, they were fixed in 3% formaldehyde in phosphate-buffered saline, and the distribution of the GFP-containing fusion proteins was analyzed on a Zeiss LSM510 META confocal microscope using a 63x 1.4-numerical aperture oil immersion planapochromal lens. For staining of nuclear pore complexes (NPC), cells were additionally permeabilized with 0.5% Triton X-100, stained with monoclonal antibody (MAb) 414 antibodies (Covance Innovative Antibodies) and AlexaFluor 546-labeled secondary antibodies, and analyzed on a confocal microscope.

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Mutations in VEEV capsid protein affect its ability to cause both CPE and cellular-transcription inhibition. In our previous work, we expressed the VEEV capsid protein from the noncytopathic VEEV replicons with a mutation in nsP2 that decreased the replication and transcription rates of virus-specific RNAs (12). Capsid protein expression from these cassettes caused rapid CPE development and inhibited RNA polymerase I- and II-dependent cellular transcription. However, fewer than 0.1% of the cells continued to grow, and their growth correlated with an accumulation of mutations in the replicon-encoded capsid gene. The majority of mutations destroyed the open reading frame downstream of aa 50 of the capsid-coding sequence, but others changed either a single amino acid (K₅₁E or Q₅₂P) or a short peptide between aa 57 and 86 (aa 58 to 85 frameshift). However, these point and frameshift mutations were detected only in the capsid gene (mutC_VEE) that encoded the protein with inactive protease (S₂₂₆A mutant). Therefore, it was not clear whether the K₅₁E, Q₅₂P, and frameshift mutations strongly affected the ability of the capsid protein to cause CPE or if this was a synergistic effect of these mutations and inactivation of protease activity by the S₂₂₆A replacement. To distinguish between these two possibilities and to further evaluate the effects of the mutations on C_VEE function, we initially cloned the above-described K₅₁E, Q₅₂P, and frameshift mutations into a VEErepL/mutC_VEE/Pac replicon, in which the capsid gene encoded a protease mutant, and into a similar replicon that encoded a wild-type capsid protein, VEErepL/C_VEE/Pac (Fig. 1A and B). All of the replicons were synthesized in vitro, and equal amounts of RNAs were transfected into BHK-21 cells. Replicons encoding GFP or capsid proteins without the indicated adaptive mutations were used as controls. We examined the cytopathogenicities of the expressed capsid proteins by evaluating the number of Pur^r foci formed per µg of transfected RNA (Fig. 1A and B) and by measuring the growth of the Pur^r cells (Fig. 1C and D) (see Materials and Methods for details). We also assessed the abilities of the expressed capsid proteins to inhibit cellular transcription (Fig. 1E and F).

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FIG. 1. Effects of capsid protein mutant expression on cellular transcription and cell growth. (A and B) Schematic representation of VEEV replicons expressing GFP or mutated capsid proteins. The arrows indicate the positions of the subgenomic promoters. Replicons expressed either CVEE with mutated protease (mutCVEE) (A) or CVEE having no mutations in the protease domain (B). VEErepL/CVEEfrsh/Pac was included in the experiments presented in panels B, D, and F as an additional control. Cells were electroporated by 5 µg of the in vitro-synthesized RNAs. Different dilutions of the electroporated cells were seeded into 100-mm tissue culture dishes. Puromycin selection was performed as described in Materials and Methods. Purr cell colonies were stained with crystal violet on days 4 to 9 posttransfection, depending on their growth rates. The results are presented in CFU per µg of RNA used for transfection. The ranges indicate variations between different experiments. (C and D) Analysis of cell growth after transfection with VEEV replicons expressing GFP and different capsid proteins. Equal numbers of electroporated cells were seeded into six-well Costar plates. Puromycin selection (10 µg/ml) was performed between 6 and 48 h posttransfection. Then, the cells were incubated in puromycin-free media, and viable cells were counted at the indicated times. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (E and F) Analysis of cellular transcription. RNA labeling in the electroporated cells was performed for 2 h with [3H]uridine at 24 h posttransfection. RNA samples were isolated and analyzed by gel electrophoresis under the conditions described in Materials and Methods. Autoradiographs of the gels are presented. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. For quantitative analysis, aliquots of the RNA samples derived from equal numbers of cells were washed on Whatman 3MM filters with trichloroacetic acid, as described in Materials and Methods, and the radioactivity was measured by liquid scintillation counting. One of two reproducible experiments is presented.

All of the mutations previously identified in the mutC_VEE gene (K₅₁E, Q₅₂P, and aa 58 to 85 frameshift) made the expressed capsid proteins noncytopathic, regardless of their presence in C_VEE or mutC_VEE. These mutations also made the capsid proteins incapable of inhibiting cellular transcription (Fig. 1E and F). In repeated experiments, cells containing replicons driving the expression of the capsid mutants demonstrated the same growth rates as did untransfected controls or cells containing VEErepL/GFP/Pac. These data unambiguously demonstrated that C_VEE gene-encoded protease activity was not involved in CPE development or downregulation of cellular transcription but that these phenomena were most likely the function of the N-terminal capsid-specific peptide (aa 1 to 110), previously identified as a domain functioning in RNA packaging and nucleocapsid formation.

Deletion analysis of the VEEV capsid protein. The alphavirus capsid protein was shown to contain two structural domains. The C-terminal domain expresses a protease activity required for cotranslational self-cleavage of the capsid from the structural polyprotein (19, 20). The N-terminal domain is highly positively charged (40) and functions in the packaging of the viral genome during core assembly (43). This domain is known to be unfolded (7), except for a short peptide that is predicted to form an alpha helix (helix I) and is present in the capsid proteins of all of the alphaviruses (32, 33). The data presented above and the results of our previous work (12) strongly indicated that the N-terminal, but not the C-terminal, domain determines C_VEE function in CPE development and inhibition of cellular transcription. Therefore, to identify a particular peptide that exhibits these inhibitory effects, we performed a detailed deletion analysis, in which different N-terminal C_VEE fragments were expressed as GFP fusions from the VEErepL replicon. The use of GFP-tagged fragments allowed us not only to follow changes in the intracellular distributions of the proteins, but to mimic, to some extent, the natural structure of the original C_VEE, because, like the VEEV capsid C-terminal domain, GFP folds into a globular structure. In addition, the designed fusion proteins had molecular weights similar to that of C_VEE.

To generate data comparable with previous results, fusions were expressed from the VEErepL replicon. Both the C_VEE110-GFP fusion, which expressed the entire N-terminal domain, and C_VEE80-GFP (Fig. 2A) with the highly positively charged fragment deleted were very efficient in causing both cell death (Fig. 2B) and inhibition of cellular transcription (Fig. 2C), and very few replicon-containing cells developed Pur^r foci (Fig. 2A). These data indicated that the critical peptide was located upstream of the deletions made.

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FIG. 2. The N-terminal CVEE fragment inhibits cellular transcription and causes CPE development. (A) Schematic representation of VEEV genome-based replicons expressing the amino-terminal fragments of CVEE fused with GFP and analysis of their abilities to establish persistent replication and develop Purr foci. The arrows indicate the positions of the subgenomic promoters. The numbers indicate the last capsid-specific amino acids in fusion proteins. (B) Analysis of cell growth after transfection with VEEV replicons expressing GFP or the indicated fusions. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (C) Inhibition of transcription in the BHK-21 cells transfected with VEEV replicons expressing the indicated proteins. RNA labeling with [3H]uridine was performed for 2 h at 24 h posttransfection. Electroporation, RNA labeling, and analysis were performed as described in the legend to Fig. 1 and in Materials and Methods. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. Quantitative analysis of residual cellular transcription was performed as indicated in the legend to Fig. 1. The constructs presented were analyzed in the same set of experiments as the expression cassettes presented in Fig. 4. Therefore, the same controls were used.

The next set of deletions was designed based on the alignment of the N-terminal C_VEE fragment with that of other alphaviruses. A large number of the amino acids in the 34-to-68 peptide of C_VEE are identical to those in the corresponding fragments of other New World alphaviruses, i.e., EEEV and WEEV, but not in the Old World alphaviruses (whose capsid proteins are not cytopathic) (Fig. 3A). This finding suggested that this peptide might determine the activities of the New World alphavirus capsid proteins in transcription inhibition. Therefore, we fused the sequence of the C_VEE68 peptide, encoding the N-terminal peptide with the entire conserved region, with GFP and expressed it from the VEErepL replicon (Fig. 3B shows the details). Other constructs had either partial deletion of conserved sequence (C_VEE60-GFP) or deletion of the entire conserved peptide downstream of aa 33 (C_VEE33-GFP). The results were in agreement with the assumption about the critical role of the aa 34 to 68 peptide in virus-host cell interactions: the expression of the C_VEE68-GFP fusion caused CPE (Fig. 3C) and, by 24 h posttransfection, downregulated cellular transcription to an almost undetectable level (Fig. 3D). C_VEE60 and C_VEE33 were incapable of causing transcriptional shutoff, and GFP-tagged peptides were as noncytopathic as GFP itself. Taken together, these data indicated that a protein fragment located upstream of aa 68 is critically involved in the inhibition of cellular transcription and CPE development.

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FIG. 3. Analysis of the effects of the N-terminal VEEV capsid fragment on cellular transcription and cell viability. (A) Sequence alignment of CVEE30-68 peptide with the corresponding capsid fragments of other alphaviruses: VEEV (25), EEEV (45), SINV, and SFV (44). Helix I sequences are indicated in red. Residues identical to those in the VEEV sequence are indicated by dashes. The stars indicate the positions of the deletions introduced for better alignment of the sequences. (B) Schematic representation of VEEV genome-based replicons expressing the amino-terminal fragments of CVEE fused with GFP and analysis of their abilities to establish persistent replication and to develop Purr foci. The arrows indicate the positions of the subgenomic promoters. The numbers indicate the last capsid-specific amino acids in fusion proteins. The results are presented in CFU per µg of RNA used for transfection. The ranges indicate variations between the experiments. (C) Analysis of cell growth after transfection with VEEV replicons expressing GFP and the indicated fusions. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (D) Analysis of cellular transcription. RNA labeling with [3H]uridine was performed for 2 h at 24 h posttransfection. RNA samples were analyzed by gel electrophoresis under the conditions described in the legend to Fig. 1 and Materials and Methods. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. Quantitative analysis of residual cellular transcription was performed as indicated in the legend to Fig. 1.

To define the beginning of the critical peptide, the C_VEE deletion mutants were designed in the context of complete C_VEE-GFP fusion. Therefore, initial experiments were aimed at demonstrating that such fusion has exactly the same functions in modification of cell biology as C_VEE itself. The last amino acid in the capsid sequence was deleted to avoid cleavage of the fusion by capsid-associated protease, and fusion with GFP was performed through a flexible peptide (see Materials and Methods for details). The in vitro-synthesized VEErepL/C_VEE-GFP/Pac and control replicons VEErepL/C_VEE/Pac and VEErepL/GFP/Pac (Fig. 4A) were transfected into BHK-21 cells. C_VEE-GFP-expressing cells developed CPE at exactly the same rate as those expressing C_VEE (Fig. 4B) and demonstrated the same inhibition of cellular transcription (Fig. 4C). The mutation introduced into the C terminus abolished cleavage, and therefore, the detected phenotype could not be explained by partial processing (Fig. 4D) and C_VEE release. No cleavage was detected even by using the high-sensitivity settings on the infrared imager (data not shown).

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FIG. 4. Comparative analysis of the effects of CVEE and a CVEE-GFP fusion on cellular transcription and cell viability. (A) Schematic representation of VEEV genome-based replicons expressing CVEE and a CVEE-GFP fusion and analysis of the replicons' abilities to establish persistent replication and develop Purr foci. The arrows indicate the positions of the subgenomic promoters. (B) Analysis of cell growth after transfection with VEEV replicons expressing either GFP or the indicated capsid variants. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (C) Analysis of cellular transcription. RNA labeling with [3H]uridine was performed for 2 h at 24 h posttransfection. Electroporation, RNA labeling, and analysis were performed as described in the legend to Fig. 1 and in Materials and Methods. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. Quantitative analysis of residual cellular transcription was performed as indicated in the legend to Fig. 1. (D) Analysis of GFP and CVEE-GFP expression by the indicated replicons. Cell lysates were prepared at 20 h posttransfection and analyzed by Western blotting using GFP-specific antibody and infrared dye 800CW-labeled secondary antibodies. The images were acquired on an Odyssey Infrared Imager (LI-COR).

In the following experiments, we deleted (i) the fragment encoding the peptide upstream of helix I (aa 2 to 31), (ii) the upstream fragment and a part of helix I (aa 2 to 40), and (iii) helix I only (aa 35 to 47) (Fig. 5A). The first deletion did not abolish the ability of the fusion protein to cause CPE; however, the deletions of aa 2 to 40 and 35 to 47 made fusion proteins incapable of both CPE induction (Fig. 5B) and cellular-transcription inhibition (data not shown). Thus, the results strongly indicated that the entire helix I is required for the capsid protein to be active in these processes.

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FIG. 5. Analysis of the effects of the N-terminal deletions in capsid protein on cellular transcription and cell viability. (A) Schematic representation of VEEV genome-based replicons expressing the deleted forms of capsid protein fused with GFP and analysis of their abilities to establish persistent replication and develop Purr foci. The arrows indicate the positions of the subgenomic promoters. The numbers indicate the first amino acids of CVEE after deletion. In all of the constructs, the initiating capsid protein-specific AUG was present in its natural position. (B) Analysis of the growth of the cells carrying VEEV replicons expressing GFP or the indicated fusions. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations.

To confirm the conclusion that the C_VEE34-68 peptide can function as the entire capsid protein in causing CPE and cellular-transcription inhibition, we designed a cassette in which the GFP gene was fused with this minimal C_VEE30-68-coding sequence. Four amino acids (aa 30 to 33) were left upstream of helix I to preserve its proper folding. An AUG codon was cloned upstream of the C_VEE sequence, and fusion with GFP was performed through a flexible linker (see Materials and Methods for details). The VEErepL/C_VEE30-68-GFP/Pac replicon was as cytopathic as VEErepL/C_VEE-GFP/Pac (Fig. 6A and B), and the replicon expressing a shorter C_VEE30-60-GFP version of the protein did not noticeably affect cell growth and cellular RNA synthesis (Fig. 6B and C). This finding could potentially be explained by the deletion of the computer-predicted nuclear localization signal (NLS) (aa 64 to 68) in the C_VEE30-60 peptide, which could lead to a less efficient translocation of the protein to the nucleus. To study this possibility, we cloned three standard simian virus 40 (SV40) T-antigen (TAg) NLSs into the C terminus of the GFP. This modification strongly increased the concentration of the protein (C_VEE30-60-GFP_NLS) in the cell nuclei (Fig. 6D) but did not make the constructs cytopathic (Fig. 6B and C). To rule out the possibility that three NLSs had an effect on fusion protein functioning in transcription inhibition and CPE development, this sequence was cloned into other fusions (C_VEE-GFP_NLS and C_VEE30-68-GFP_NLS in Fig. 6A), and these additional NLSs did not have any detectable effect on protein functions (Fig. 6B and C). Thus, the inability of C_VEE30-60-GFP protein to inhibit cellular transcription was not a result (or at least not the only result) of modification of its compartmentalization, but rather was due to the loss of this peptide's ability to cause transcriptional shutoff and, ultimately, CPE development.

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FIG. 6. Effects of the CVEE peptides fused with either GFP or GFPNLS on cell viability and cellular transcription. (A) Schematic representation of VEEV genome-based replicons expressing the deleted forms of capsid protein fused with GFP and analysis of their abilities to establish persistent replication and to develop Purr foci. The arrows indicate the positions of the subgenomic promoters. In all of the constructs, the initiating AUG was created upstream of the studied peptide. (B) Analysis of cell growth after transfection of VEEV replicons encoding the indicated fusions. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (C) Analysis of cellular transcription. RNA labeling with [3H]uridine was performed for 2 h at 24 h posttransfection. Electroporation, RNA labeling, and analysis were performed as described in the legend to Fig. 1 and in Materials and Methods. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. Quantitative analysis of residual cellular transcription was performed as indicated in the legend to Fig. 1. (D) Intracellular distribution of CVEE30-60-GFP and CVEE30-60-GFPNLS fusions. BHK-21 cells were transfected with the corresponding replicons, and the intracellular distributions of the fusions were analyzed at 12 h posttransfection on an inverted fluorescence microscope (Leica).

Taken together, the results of this study strongly indicated that the peptide located between aa 33 and 69 of C_VEE is critically involved in CPE development and transcription inhibition. Both helix I and the downstream sequence (aa 52 to 68) are required for the capsid protein activities in these processes. The deletions in helix I and in the downstream peptide made C_VEE30-68 nonfunctional.

The EEEV-specific, helix I-containing peptide functions similarly to its VEEV-specific counterpart. As we described above, the C_VEE30-68 peptide demonstrates a significant level of conservation among the New World alphaviruses (Fig. 3A). Therefore, it is reasonable to expect that these peptides have similar functions in EEEV and WEEV. To test this hypothesis, we expressed aa 33 to 71 of C_EEE (homologous to aa 30 to 68 of C_VEE) as a GFP fusion from a VEErepL replicon (Fig. 7A). In our research, C_EEE was not studied as intensively as C_VEE, and we did not have the exact information establishing that the NLS(s) required for its intranuclear function is located in this particular peptide or in different amino-terminal sequences of the protein. Therefore, to demonstrate the activity of the C_EEE33-71-GFP fusion in cellular-transcription inhibition and CPE development, three additional SV40 TAg NLSs were cloned into the carboxy terminus of GFP (Fig. 7A). After transfection of the in vitro-synthesized RNAs, cells expressing the C_EEE33-71-GFP_NLS fusion demonstrated the same rates of CPE development as did those with C_VEE30-68-GFP_NLS (Fig. 7B), and transcription in these cells was inhibited to a similar level (Fig. 7C).

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FIG. 7. Comparative analysis of the effects of CVEE30-68 and CEEE33-71 peptides fused with GFPNLS on cellular transcription and cell viability. (A) Schematic representation of VEEV genome-based replicons expressing GFP or fusion proteins and analysis of the replicons' abilities to establish persistent replication and develop Purr foci. The initiating AUG was created upstream of the studied peptides. (B) Analysis of cell growth after transfection with VEEV replicons expressing the indicated proteins. The data were normalized on the number of viable, adherent cells determined at 6 h posttransfection. The error bars indicate standard deviations. (C) Analysis of cellular transcription. RNA labeling with [3H]uridine was performed for 2 h at 24 h posttransfection. Electroporation, RNA labeling, and analysis were performed as described in the legend to Fig. 1 and in Materials and Methods. The positions of the ribosomal 28S and 18S and preribosomal 45S RNAs are indicated. Quantitative analysis of residual cellular transcription was performed as indicated in the legend to Fig. 1.

This result indicated that the ability to interfere with the transcription of cellular RNAs is a feature of the same peptide in capsid proteins derived from two different New World alphaviruses, and the latter group of viruses appears to employ similar mechanisms of interference with cellular gene expression.

Intracellular distribution of the VEEV capsid protein. As we demonstrated in the previous study, C_VEE is distributed not only in the cytoplasm, but also in the nuclei of virus-infected cells (12). Its presence in the nucleus might be determined by a combination of active nuclear import and passive diffusion. Based on computer predictions, the VEEV capsid protein appears to contain a number of NLS-like sequences that might promote its transport into the nucleus, and the majority of these putative signals are concentrated within the peptide between aa 57 and 86, which in our previous study was changed by two attenuating frameshift mutations (12) (VEErepL/C_VEEfrsh/Pac in Fig. 1 shows the details). These data indicated that the putative NLS(s) might be functional and important for C_VEE activity in the inhibition of transcription. To further test this hypothesis, we compared the intracellular distribution of C_VEE-GFP and C_VEEfrsh-GFP fusions expressed by VEErepL replicons. These fusion proteins are too large for efficient diffusion through the nuclear pores (30), and their presence in the nucleus should be mainly determined by the NLS-dependent active transport. The results presented in Fig. 8A and B demonstrate that a significant fraction of C_VEE-GFP was transported into the nucleus and that the aa 58 to 85 frameshift blocked the ability of the protein to accumulate in the cell nuclei. These data strongly indicated that the peptide located between aa 58 and 85 of C_VEE contains an NLS(s) that functions in the nuclear cytoplasmic traffic.

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FIG. 8. Intracellular distributions of different CVEE-GFP fusions. (A and B) BHK-21 cells were transfected with the replicons expressing CVEE-GFP (a), CVEE35-47-GFP (b), and CVEEfrsh-GFP (c) proteins, and the intracellular distributions of the fusions were analyzed at 12 h posttransfection. The images were acquired on a confocal microscope. Different magnifications are presented in panels A and B. (C) BHK-21 cells were transfected with VEErepL/CVEE-GFP/Pac, and, at 12 h posttransfection, the cells were permeabelized with 0.5% Triton X-100, stained with MAb 414 antibody and AlexaFluor 546-labeled secondary antibodies, and analyzed on a confocal microscope. (a) Distribution of CVEE/GFP on the nuclear membrane. (b) MAb 414 staining of the same cell. (c) Overlay of the images (the enlarged fragment is indicated in a and b). The bars in panel A correspond to 20 µm, and in panels B and C, to 5 µm.

The presence of an active NLS(s) in the capsid protein raised an interesting question: why does this protein not completely concentrate in the nucleus? Its presence in the cytoplasm might be explained by binding to ribosomes and/or sequestration during core assembly; however, the intracellular distribution of another C_VEE-GFP mutant led us to an alternative explanation. The C_VEE-GFP fusion, which had helix I deleted (VEErepL/C_VEE35-47-GFP/Pac replicon in Fig. 5A), accumulated only in the cell nuclei (Fig. 8A and B). This was an indication that helix I either (i) is required for binding of the capsid protein to cytoplasmic factors, (ii) stimulates an immediate assembly of capsid molecules into higher-order core structures, or (iii) functions as a nuclear export signal supporting the balance between the nuclear and cytoplasmic forms of capsid protein.

A more detailed analysis of C_VEE-GFP compartmentalization revealed another interesting feature of this protein. Significant fractions of C_VEE-GFP (Fig. 8A, B, and C) and C_VEE30-68-GFP (data not shown) fusions were detected in the nuclear rim, where both proteins demonstrated a punctate pattern of distribution reminiscent of that of the NPCs. Cells expressing C_VEE-GFP were additionally stained with nuclear pore-specific antibody (MAb 414), which recognizes the conserved FXFG repeats in several nucleoporins, namely, Nup62, Nup98, Nup153, Nup214, and Nup358. By using confocal microscopy, we demonstrated a strong colocalization of C_VEE-GFP with the MAb 414-stained NPCs. (Fig. 8C). We also detected in the cytoplasm some large C_VEE-GFP-containing complexes that were also capable of MAb 414 binding (data not shown). We speculate that these structures are the annulate lamellae (22) that contain nuclear-pore-like complexes. Thus, our results indicate that the wild-type capsid protein and its C_VEE30-68 variant might be capable of interacting with NPC and thus modifying the nuclear cytoplasmic trafficking. Alterations of helix I or replacement of the helix I-containing fragment by that of SINV capsid protein (data not shown) abolished localization of fusion proteins on the nuclear membrane.

Attenuated VEEV variants. As we indicated above, C_VEE expression by VEErepL determined the development of CPE and transcriptional shutoff in cells of vertebrate origin. However, the capsid protein of another alphavirus, SINV, was incapable of causing both phenomena. These results suggested that VEEV might be attenuated by making the mutations in the critical C34-68 peptide or by replacing this peptide with the corresponding C_SIN-derived fragment. Two of the possible variants (Fig. 9A) were designed on the basis of the VEEV TC-83 genome. The original TC-83 strain is attenuated for adult mice but is lethal for 6-day-old mice after i.c. inoculation (31). Therefore, this virus can be used for making additional genetic manipulations and analyses of their effects on pathogenesis. In VEEV/C_SIN1, the natural VEEV capsid gene was replaced by a chimeric version, in which the entire N-terminal fragment, located upstream of the protease domain (aa 1 to 110), was replaced by its SINV-specific counterpart (aa 1 to 98). The second variant, VEEV/Cfrsh, encoded the capsid protein with the above-described frameshift mutations that changed the peptide between aa 57 and 86. It should be noted that this frameshift did not change the highly hydrophilic nature of the peptide and even increased the number of positively charged amino acids in the amino terminus. However, as we described above, the frameshift affected the ability of C_VEE to translocate to the nucleus. Both in vitro-synthesized, mutant genomes were as infectious as the control VEEV TC-83 RNA and generated homogeneous plaques in the infectious-center assay (data not shown), indicating that no additional mutations were required for virus viability. In BHK-21 cells, both viruses demonstrated growth rates comparable to those of VEEV TC-83 (Fig. 9B); however, in contrast to TC-83, they became noncytopathic and did not prevent cell growth in the medium with 10% FBS. Nevertheless, they were still capable of forming plaques in BHK-21 cells under agarose cover with a low serum concentration.

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FIG. 9. Replication of VEEV TC-83 with mutated capsid protein. (A) Schematic representation of the viral genomes. In VEEV/CSIN1, the natural N-terminal fragment, located upstream of the protease domain (aa 1 to 110), was replaced by its SINV-specific counterpart (aa 1 to 98), indicated by a black box. In VEEV/Cfrsh, the capsid protein gene contained a frameshift mutation that changed the peptide between aa 57 and 86. (B) BHK-21 cells were electroporated with 5 µg of in vitro-synthesized RNAs. One-fifth of the samples were seeded into 35-mm culture dishes. At the indicated times posttransfection, the media were replaced by fresh media, and virus titers in the culture fluids were determined by a plaque assay on BHK-21 cells. Note that cells transfected with VEEV/CSIN1 and VEEV/Cfrsh RNAs continued to release virus after 24 h posttransfection, when VEEV TC-83 RNA-transfected cells developed a profound CPE. (C) Survival of mice infected with the indicated viruses. Six-day-old NIH Swiss mice were inoculated i.c. with the indicated doses of viruses. The animals were monitored for 2 months. No deaths occurred after day 9 postinfection in any of these experiments.

The mutant viruses were further tested for their pathogenicity in suckling mice. Animals were i.c. inoculated with 10⁶ to 10⁷ PFU of VEEV/C_SIN1, VEEV/Cfrsh, and two comparable doses of VEEV TC-83 (Fig. 9C). Both doses of the last virus were universally lethal for mice of this age. VEEV/C_SIN1 and VEEV/Cfrsh caused death in only a fraction of the mice, and both capsid mutants yielded significantly more mean survival days in the infected mice than the VEEV TC-83 parent (Student's t test; P < 0.001). Thus, modification of C_VEE by replacing the amino-terminal fragment with the SINV-specific counterpart or by a frameshift mutation affecting the C_VEE intracellular distribution caused additional VEEV TC-83 attenuation. These data demonstrated the importance of C_VEE and the critical peptide, C_VEE34-68, in particular for virus replication and pathogenesis. The surviving mice exhibited the presence of neutralizing antibodies in their sera and were protected against future infection with an enzootic strain of VEEV (data not shown).

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The natural transmission cycle of VEEV and other alphaviruses is based on the development of high-titer viremia in vertebrate hosts, which is required for infection of mosquitoes during the blood meal. The level of viremia and its duration are the critical factors that determine the successful continuation of the enzootic cycle and virus persistence in nature. However, like any other viral infection, alphavirus replication in vertebrate cells induces a response aimed at downregulation of virus production and activation of cell signaling. This leads to activation of the antiviral state in the uninfected cells and prevention of successive rounds of infection. As do most, if not all, other viruses, alphaviruses have developed efficient mechanisms of interference with the cellular response, and one of them is based on the inhibition of cellular transcription (9, 13). In the Old World alphaviruses SINV and SFV, the nonstructural protein nsP2 is a key player in the inhibition of transcription of cellular mRNA and rRNA. However, the New World alphaviruses VEEV and EEEV employ not nsP2, but the structural capsid protein to achieve the same goal. The capsid protein is efficiently expressed in the infected cells and distributed in both the cytoplasm and nuclei. Besides packaging viral genome RNA, VEEV- and EEEV-specific capsid proteins cause global inhibition of cellular transcription that strongly correlates with CPE development. The results of this study demonstrate that both phenomena are determined by the same short, N-terminal peptide of C_VEE, positioned between aa 33 and 69. We speculate that CPE development is a result of the transcriptional block; however, the possibility that these effects are determined by different mechanisms cannot be completely ruled out yet.

The identified functional capsid peptide can be provisionally divided into two small domains: (i) a previously defined -helix, helix I (aa 34 to 51) (32, 33), that is present in all of the alphavirus capsid proteins and has been shown to function in core assembly and (ii) a downstream, highly positively charged peptide located between aa 51 and 69. The amino acid sequences of both domains demonstrate strong conservation among the New World alphaviruses, but these peptides differ from the sequences in the Old World alphavirus capsid proteins, which are nonfunctional in transcription inhibition and incapable of causing CPE. Alterations of each domain in C_VEE34-68 have strong negative effects on the ability of the entire C_VEE or minimal C_VEE30-68 peptide to cause transcriptional shutoff and CPE. However, these mutations appear to affect different functions. A C_VEE-GFP fusion lacking the helix I domain (C_VEE35-47-GFP) accumulated only in the cell nuclei, and C_VEEfrsh, with frameshift mutations in the downstream peptide (aa 58 to 85), was no longer transported to the nucleus. These mutations changed an aa 58 to 85 sequence in a very interesting way: the peptide remained highly hydrophilic and became even more positively charged. However, based on the computer prediction, the putative NLSs located between aa 64 and 85 were destroyed. The C_VEE-GFP fusion protein is larger than proteins that can passively diffuse to the nucleus, but in our experiments, a significant fraction of this protein was found in that compartment. Since the frameshift mutation made the fusion protein incapable of translocation to the nuclei, this finding was a strong indication that the NLS(s) predicted after aa 64 is indeed functional and drives C_VEE-GFP and most likely C_VEE itself into the nucleus.

The increase in nuclear localization of C_VEE after the helix I deletion might be explained in different ways. First, as previously described for C_SIN (32, 33), the helix I deletion might strongly affect the nucleocapsid assembly and consequently the balance between the core-associated capsid protein and its free form, which, as we indicated above, is capable of translocating to the nucleus. Another explanation is that, in addition to another previously proposed peptide (51) functioning in C_SIN binding to the ribosomes, VEEV helix I is involved in this function and thus in retention of C_VEE in the cytoplasm. Finally, C_VEE helix I might function as a nuclear export signal, and the balance between C_VEE import to the nucleus and its export determines its intracellular distribution. Because these data are important for a further understanding of C_VEE functioning in virus-host cell interactions, all of these possibilities need experimental testing in future studies.

The most important finding, however, was not the changes in C_VEE compartmentalization due to the helix I deletion or mutations in aa 58 to 85, but the detected inability of the mutated proteins to inhibit cellular transcription and cause CPE. (i) Accumulation of the helix I deletion mutant of capsid protein in the nucleus did not noticeably affect cell biology and strongly suggested that helix I not only functions in the core assembly and control of intracellular distribution of the capsid but appears to also be directly or indirectly involved in the development of transcriptional shutoff. (ii) The C_VEE52-68 domain might contain a functional NLS at the end of the peptide (which is deleted in the nonfunctional C_VEE30-60 peptide). However, the addition of the efficient three SV40 TAg NLSs, which led to complete translocation of C_VEE30-60-GFP_NLS into the nucleus, did not make this protein as functional as C_VEE30-68-GFP in the inhibition of cellular transcription. Moreover, point mutations in aa 51 and 52 (outside the putative NLS) also made C_VEE incapable of inhibiting transcription. Taken together, the data strongly suggested that both domains, helix I and aa 52 to 68, appear to have more sophisticated functions in cellular transcription regulation than merely the control of intracellular C_VEE distribution.

One of the possible explanations for the capsid protein activity in transcription inhibition may lie in the modification of nucleocytoplasmic transport. Significant fractions of C_VEE-GFP (Fig. 8A and B) and C_VEE30-68-GFP were detected on the nuclear membrane, where they demonstrated a distribution similar to that of the NPCs, suggesting an interaction between Nups and C_VEE. To date, inhibition of nuclear transport has been described only for a very limited number of viruses, among which vesicular stomatitis virus, poliovirus, rhinovirus, and cardiovirus are better studied (16, 28, 34, 38). The VSV matrix protein interacts with the nucleoporin Nup98 and the export receptor Rae 1 (8). Thus, M protein accumulates in the NPC (35), in which it efficiently inhibits Rae 1-mediated mRNA nuclear export (35, 47) and slows the rate of nuclear import through the importin /ß1-dependent pathway (34). Interestingly, vesicular stomatitis virus also efficiently inhibits cellular transcription (4), but the correlation between inhibition of nucleocytoplasmic traffic and downregulation of transcription has not been investigated. Picornaviruses have been previously shown to alter nucleocytoplasmic transport either by the protease-dependent processing of nucleoporins (17, 18) or by disruption of the RanGDP/GTP gradient (38). Therefore, VEEV, and most likely other New World alphaviruses, join a growing number of pathogens that interfere with the activation of cellular genes that function in the antiviral response by modifying nucleocytoplasmic transport. The importance of C_VEE localization on the NPC is currently supported by two findings. (i) A significant fraction of C_SIN (the noncytopathic capsid protein) is present in the nucleus but is not associated with the nuclear membrane (data not shown), and this might be a plausible explanation for C_SIN's inability to cause transcriptional shutoff. (ii) As indicated above, C_VEE with a deleted helix I sequence is present in the nucleus at a high concentration; however, it does not accumulate on the nuclear membrane/nuclear pores, and this strongly correlates with its inability to inhibit transcription. Moreover, our preliminary experiments demonstrated that C_VEE inhibits at least one nuclear import pathway that is mediated by the importin-/ß receptors (data not shown) and prevents translocation of the SV40 NLS-containing proteins to the nuclei. The exact mechanism of C_VEE-specific NPC modification and its relationship with transcriptional shutoff are now under intensive investigation in our laboratories.

The importance of the newly identified functions of C_VEE for virus replication was strongly supported by in vitro and in vivo experiments with replicating VEEV TC-83, encoding modified versions of the capsid protein. The replacement of the entire amino-terminal fragment of C_VEE with that of C_SIN in VEEV/C_sin1 did not noticeably change the replication of the virus in BHK-21 cells, and the original VEEV TC-83 and VEEV/C_sin1 demonstrated nearly identical growth rates. However, the mutated virus was dramatically less cytopathic, and cells continued to release infectious virus for days (after complete CPE development in VEEV TC-83-infected cells). Moreover, this mutant was more attenuated in vivo than the currently available vaccine strain, VEEV TC-83. The introduction of the above-described frameshift mutations into the capsid gene of TC-83 had a detectable negative effect on virus replication. This lower level of virus replication might explain its more attenuated phenotype in vivo. However, its strongly altered ability to cause CPE in cultured cells points to the possibility that a change in C_VEE interactions with cellular transcriptional machinery may also be involved.

Taken together, the results of this study suggest new approaches for attenuation of the New World alphaviruses: (i) the identified critical domain of VEEV and EEEV (and probably WEEV) capsid proteins can be modified by point mutations or small deletions or (ii) the large fragments of the protein can be replaced by the Old World alphavirus-derived counterparts. The second direction is likely to prove more promising, because one of the distinguishing features of alphaviruses is in their extraordinarily high mutation and adaptation rates. Therefore, the effects of point mutations and small deletions, which have a negative effect on virus growth rates, are usually neutralized by adaptive, compensatory mutations within a few passages in vivo or in vitro. It will likely be more difficult to adapt the entire amino-terminal, SINV-specific fragment (C1 to -98) or the entire C_SIN, which has not been adapted for transcriptional shutoff during previous SINV evolution.

In conclusion, we have demonstrated the following. (i) The amino-terminal fragments of the VEEV and EEEV capsid proteins contain an 35-aa-long peptide that functions in the inhibition of cellular transcription and CPE development. (ii) The identified VEEV-specific peptide C_VEE33-68 includes two domains with distinct functions: the -helix domain, helix I, which is critically involved in supporting the balance between the presence of the protein in the cytoplasm and the nucleus, and the C-terminal peptide, which might contain a functional NLS(s). The integrity of both domains determines the intracellular distribution of C_VEE, and both are essential for capsid protein function in the inhibition of transcription. (iii) C_VEE appears to interact with NPC and, this interaction correlates with the protein's ability to cause transcriptional shutoff and, ultimately, CPE. (iv) The replacement of the N-terminal fragment of C_VEE by its SINV-specific counterpart in the VEEV TC-83 genome does not affect virus replication in vitro but strongly reduces cytopathogenicity and virulence in vivo. These findings can be used in designing a new generation of live, attenuated, recombinant vaccines against the New World alphaviruses.

 
We thank Mardelle Susman, technical editor, for critical reading and editing of the manuscript.

This work was supported by Public Health Service grant AI050537 and a grant from NIAID through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, U54 AI057156.

 
* Corresponding author. Mailing Address: Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019. Phone: (409) 772-2327. Fax: (409) 772-5065. E-mail: ivfrolov{at}UTMB.edu

Published ahead of print on 3 October 2007.

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Journal of Virology, December 2007, p. 13552-13565, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01576-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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