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

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 CVEE peptide, CVEE33-68, 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.