Elena I. Frolova

The Old World and New World Alphaviruses Use Different Virus-Specific Proteins for Induction of Transcriptional Shutoff

ABSTRACT Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.

Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection

ABSTRACT Alphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-α/β) system or when IFN activity is neutralized by anti-IFN-α/β antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.

Sindbis Virus Nonstructural Protein nsP2 Is Cytotoxic and Inhibits Cellular Transcription

ABSTRACT Replication of alphaviruses in vertebrate cells strongly affects cell physiology and ultimately leads to development of a cytopathic effect (CPE) and cell death. Sindbis virus (SIN) replication causes major changes in cellular macromolecular synthesis, in which the strong downregulation of transcription of cellular mRNAs and rRNAs plays a critical role. SIN nonstructural protein nsP2 was previously proposed as one of the main regulators of virus-host cell interactions, because point mutations in the carboxy-terminal part of nsP2 could make SIN and other alphaviruses and replicons less cytopathic and capable of persisting in some vertebrate cell lines. These mutants were incapable of inhibiting transcription and downregulating a viral stress-induced cell response. In the present work, we demonstrate that (i) SIN nsP2 is critically involved in CPE development, not only during the replication of SIN-specific RNAs, but also when this protein is expressed alone from different expression cassettes; (ii) the cytotoxic effect of SIN nsP2 appears to be at least partially determined by its ability to cause transcriptional shutoff; (iii) these functions of SIN nsP2 are determined by the integrity of the carboxy-terminal peptide of this protein located outside its helicase and protease domains, rather than by its protease activity; and (iv) the cytotoxic activity of SIN nsP2 depends on the presence of this protein in a free form, and alterations in P123 processing abolish the ability of nsP2 to cause CPE.

Inhibition of Transcription and Translation in Sindbis Virus-Infected Cells

ABSTRACT Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. The natural transmission cycle of alphaviruses requires their presence at high concentrations in the blood of amplification hosts for efficient infection of mosquito vectors. The high-titer viremia development implies multiple rounds of infection that proceed in the background of the developing antiviral cell response aimed at blocking virus spread on an organismal level. Therefore, as for many viruses, if not most of them, alphaviruses have evolved mechanisms directed toward downregulating different components of the antiviral cell reaction and increasing viremia to a level sufficient for the next round of transmission. Using Sindbis virus (SIN) as a model, we demonstrated that (i) the replication of wild-type SIN strongly affects major cellular processes, e.g., transcription and translation of mRNAs; (ii) transcriptional and translational shutoffs are distinctly independent events, and their development can be differentially manipulated by creating different mutations in SIN nonstructural protein nsP2; and (iii) inhibition of transcription, but not translation, is a critical mechanism that SIN employs to suppress the expression of cellular viral stress-inducible genes in cells of vertebrate origin. Downregulation of transcription of all of the cellular mRNAs appears to be a very efficient means of reducing the development of an antiviral response. The ability to cause transcriptional shutoff may partially determine SIN host range and replication in particular tissues.

Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53–p73 complex as a promising and highly specific potential target for cancer therapy.

Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane

ABSTRACT Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However, our recent studies have demonstrated that Sindbis virus (SINV)-specific, double-stranded RNA (dsRNA)- and nonstructural protein (nsP)-containing RCs are initially formed at the plasma membrane. In this new study, we present extensive evidence that (i) in cells of vertebrate origin, at early times postinfection, viral nsPs colocalize with spherules at the plasma membrane; (ii) viral dsRNA intermediates are packed into membrane spherules and are located in their cavities on the external surface of the plasma membrane; (iii) formation of the membrane spherules is induced by the partially processed nonstructural polyprotein P123 and nsP4, but synthesis of dsRNA is an essential prerequisite of their formation; (iv) plasma membrane-associated dsRNA and protein structures are the active sites of single-stranded RNA (ssRNA) synthesis; (v) at late times postinfection, only a small fraction of SINV nsP-containing complexes are relocalized into the cytoplasm on the endosome membrane. (vi) pharmacological drugs inhibiting different endocytotic pathways have either only minor or no negative effects on SINV RNA replication; and (vii) in mosquito cells, at any times postinfection, dsRNA/nsP complexes and spherules are associated with both endosomal/lysosomal and plasma membranes, suggesting that mechanisms of RC formation may differ in cells of insect and vertebrate origins.

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

ABSTRACT Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

Two novel 21‐residue antimicrobial peptides, arenicin‐1 and arenicin‐2, exhibiting activity against Gram‐positive and Gram‐negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP‐HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) 1 were determined for each isoform. Each arenicin has one disulfide bond (Cys3‐Cys20). The total RNA was isolated from the lugworm coelomocytes, RT‐PCR and cloning were performed, and cDNA was sequenced. A 202‐residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.

PKR-dependent and -independent mechanisms are involved in translational shutoff during Sindbis virus infection.

ABSTRACT The replication of Sindbis virus (SIN) profoundly affects the metabolism of infected vertebrate cells. One of the main events during SIN infection is the strong inhibition of translation of cellular mRNAs. In this study, we used a combination of approaches, including the study of SIN replication in PKR−/− mouse embryo fibroblasts or in the presence of an excess of catalytically inactive PKR. We show that the PKR-dependent inhibition of translation is not the only and most likely not the major pathway mediating translational shutoff during SIN infection. The PKR-independent mechanism strongly affects the translation of cellular templates, whereas translation of SIN subgenomic RNA is resistant to inhibition, and this leads to a benefit for viral replication. Our findings suggest that both PKR-dependent and non-PKR-dependent mechanisms of SIN-induced translational shutoff can be manipulated by using SIN replicons expressing mutated SIN nsP2 or kinase-defective PKR. Specifically, we show that expression of heterologous genes from SIN-based and most likely other alphavirus-based replicons can be increased by downregulating both the PKR-dependent and PKR-independent translational shutoffs.

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

ABSTRACT 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 CVEE33-68 includes two domains with distinct functions: the α-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 proteins 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.