Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

Eastern equine encephalitis virus (EEEV) causes sporadic but often severe cases of human and equine neurological disease in North America. To determine how EEEV may evade innate immune responses, we screened individual EEEV proteins for the ability to rescue the growth of a Newcastle disease virus expressing green fluorescent protein (NDV-GFP) from the antiviral effects of interferon (IFN). Only expression of the EEEV capsid facilitated NDV-GFP replication. Inhibition of the antiviral effects of IFN by the capsid appears to occur through a general inhibition of cellular gene expression. For example, the capsid inhibited the expression of several reporter genes under the control of RNA polymerase II promoters. In contrast, capsid did not inhibit expression from a T7 RNA polymerase promoter construct, suggesting that the inhibition of gene expression is specific and is not a simple manifestation of toxicity. The inhibition correlated both with capsid-induced phosphorylation of eukaryotic initiation factor 2 alpha and with capsid-mediated inhibition of cellular mRNA accumulation. Mapping analysis identified the N terminus as the region important for the inhibition of host gene expression, suggesting that this inhibition is independent of capsid protease activity. Finally, when cell lines containing EEEV replicons encoding capsid were selected, replicons consistently acquired mutations that deleted all or part of the capsid, for example, amino acids 18 to 135. Given that the amino terminus of the capsid is required to inhibit host cell gene expression, these data suggest that capsid expression from the replicons is ultimately toxic to host cells, presumably because of its ability to inhibit gene expression. Eastern equine encephalitis virus (EEEV), a mosquito-borne member of the Alphavirus genus of the Togaviridae family, is among the deadliest of the mosquito-borne viruses. In humans, the fatality rate following symptomatic infection approaches 80%, and many survivors develop crippling sequelae, such as mental retardation, convulsions, and paralysis (18, 37).EEEV possesses a single-stranded, positive-sense RNA genome of approximately 11.7 kb. The four viral nonstructural proteins (nsP1-4) are produced from the full-length genomic RNA as a polyprotein and carry out functions important for viral RNA synthesis and polyprotein processing. A second polyprotein, translated from the 26S subgenomic mRNA, gives rise to the three major structural proteins, namely, the capsid and the two envelope proteins, E1 and E2. The envelope proteins are involved in receptor recognition, virus attachment, penetration, and membrane fusion during viral entry. These proteins also cooperate with the capsid during viral assembly and release. Based on observations made with the capsids of other alphaviruses, the EEEV capsid is expected to encapsulate the viral genomic RNA, to interact with the viral glycoproteins during assembly, and to function as a protease to release itself from the nascent structural polypeptide chain (54,63,65).

Section: Discussionsupporting

confidence: 54%

Capsid Protein of Eastern Equine Encephalitis Virus Inhibits Host Cell Gene Expression

Aguilar

Weaver

Basler

2007

“…Infectious virus was recovered from the pRSV-M-null and pRSV-⌬SH/GFP cDNAs as previously described (45), with the following modifications (for cDNA pRSV-⌬SH/ GFP, only the first and second modifications apply): (i) the initial transfection of the cDNA and support plasmids was done with BHK-21 cells expressing T7 polymerase from a nonpathogenic alphavirus replicon (47); (ii) the N, P, M2-1, and L support plasmids contained an internal ribosome entry site (IRES) preceding the ORF; (iii) plasmid pc-Mopt was included in the initial transfection to ensure virion production; and (iv) infectious virus was amplified in H2-M cells. Virus derived from pRSV-⌬ was designated recWT, and virus derived from pRSV-M-null was designated M-null.…”

Section: Methodsmentioning

confidence: 99%

The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

Mitra

Baviskar

Duncan-Decocq

et al. 2012

110

An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virusinduced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virusinfected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.

“…The difference in transcription inhibition between the New and the Old World alphaviruses is particularly evident in the cells infected with the alphavirus genome-derived replicons, which express no structural proteins. VEEV and eastern equine encephalitis virus (EEEV), but not SINV and SFV, replicons readily establish persistent replication in some of the commonly used cell lines of vertebrate origin (30,38), and this is a strong indication that in the absence of capsid protein expression, inhibition of transcription in the cells infected with the New World alphavirus replicons does not reach the level that can cause cell death.…”

mentioning

confidence: 99%

Interplay of Acute and Persistent Infections Caused by Venezuelan Equine Encephalitis Virus Encoding Mutated Capsid Protein

Atasheva

Krendelchtchikova

Liopo

et al. 2010

Self Cite

Venezuelan equine encephalitis virus (VEEV) is a significant human and animal pathogen. The highlight of VEEV replication in vitro, in cells of vertebrate origin, is the rapid development of cytopathic effect (CPE), which is strongly dependent upon the expression of viral capsid protein. Besides being an integral part of virions, the latter protein is capable of (i) binding both the nuclear import and nuclear export receptors, (ii) accumulating in the nuclear pore complexes, (iii) inhibiting nucleocytoplasmic trafficking, and (iv) inhibiting transcription of cellular ribosomal and messenger RNAs. Using our knowledge of the mechanism of VEEV capsid protein function in these processes, we designed VEEV variants containing combinations of mutations in the capsid-coding sequences. These mutations made VEEV dramatically less cytopathic but had no effect on infectious virus production. In cell lines that have defects in type I interferon (IFN) signaling, the capsid mutants demonstrated very efficient persistent replication. In other cells, which have no defects in IFN production or signaling, the same mutants were capable of inducing a long-term antiviral state, downregulating virus replication to an almost undetectable level. However, ultimately, these cells also developed a persistent infection, characterized by continuous virus replication and beta IFN (IFN-␤) release. The results of this study demonstrate that the long-term cellular antiviral state is determined by the synergistic effects of type I IFN signaling and the antiviral reaction induced by replicating viral RNA and/or the expression of VEEV-specific proteins. The designed mutants represent an important model for studying the mechanisms of cell interference with VEEV replication and development of persistent infection.