The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.

Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-β, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5’UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5’UTR, which increase the efficiency of the promoter located in the 5’end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5’UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

Hendra virus (HeV) and Nipah virus (NiV) are closely related, recently emerged paramyxoviruses that form Henipavirus genus and are capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. However, in contrast to many other species and despite expression of functional virus entry receptors, mice are resistant to henipavirus infection. We report here the susceptibility of mice deleted for the type I interferon receptor (IFNAR-KO) to both HeV and NiV. Intraperitoneally infected mice developed fatal encephalitis, with pathology and immunohistochemical features similar to what was found in humans. Viral RNA was found in the majority of analyzed organs, and sublethally infected animals developed virus-specific neutralizing antibodies. Altogether, these results reveal IFNAR-KO mice as a new small animal model to study HeV and NiV pathogenesis, prophylaxis, and treatment and suggest the critical role of type I interferon signaling in the control of henipavirus infection.

The aberrant expression of human endogenous retrovirus (HERV) elements of the HERV-W family has been associated with different diseases, including multiple sclerosis (MS). In particular, the expression of the envelope protein (Env) from the multiple sclerosis-associated retrovirus (MSRV), a member of HERV-W family and known for its potent proinflammatory activity, is repeatedly detected in the brain lesions and blood of MS patients. Furthermore, human herpesvirus 6 (HHV-6) infection has long been suspected to play a role in the pathogenesis of MS and neuroinflammation. We show here that both HHV-6A and stimulation of its receptor, transmembrane glycoprotein CD46, induce the expression of MSRV-Env. The engagement of extracellular domains SCR3 and SCR4 of CD46-Cyt1 isoform was required for MSRV-env transactivation, limiting thus the MSRV-Env induction to the CD46 ligands binding these domains, including C3b component of complement, specific monoclonal antibodies, and both infectious and UV-inactivated HHV-6A, but neither HHV-6B nor measles virus vaccine strain. Induction of MSRV-Env required CD46 Cyt-1 singling and was abolished by the inhibitors of protein kinase C. Finally, both membrane-expressed and secreted MSRV-Env trigger TLR4 signaling, displaying thus a proinflammatory potential, characteristic for this viral protein. These data expand the specter of HHV-6A effects in the modulation of the immune response and support the hypothesis that cross-talks between exogenous and endogenous viruses may contribute to inflammatory diseases and participate in neuroinflammation. Furthermore, they reveal a new function of CD46, known as an inhibitor of complement activation and receptor for several pathogens, in transactivation of HERV env genes, which may play an important role in the pathogenesis of inflammatory diseases.

Human herpesvirus 6 (HHV-6) is widely spread in the human population and has been associated with several neuroinflammatory diseases, including multiple sclerosis. To develop a small-animal model of HHV-6 infection, we analyzed the susceptibility of several lines of transgenic mice expressing human CD46, identified as a receptor for HHV-6. We showed that HHV-6A (GS) infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice, while HHV-6B (Z29) infection was inefficient. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the nontransgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Persistence in the brain was observed with infectious but not heat-inactivated HHV-6A. Immunohistological studies revealed the presence of infiltrating lymphocytes in periventricular areas of the brain of HHV-6A-infected mice. Furthermore, HHV-6A stimulated the production of a panel of proinflammatory chemokines in primary brain glial cultures, including CCL2, CCL5, and CXCL10, and induced the expression of CCL5 in the brains of HHV-6A-infected mice. HHV-6A-induced production of chemokines in the primary glial cultures was dependent on the stimulation of toll-like receptor 9 (TLR9). Finally, HHV-6A induced signaling through human TLR9 as well, extending observations from the murine model to human infection. Altogether, this study presents a first murine model for HHV-6A-induced brain infection and suggests a role for TLR9 in the HHV-6A-initiated production of proinflammatory chemokines in the brain, opening novel perspectives for the study of virus-associated neuropathology. IMPORTANCEHHV-6 infection has been related to neuroinflammatory diseases; however, the lack of a suitable small-animal infection model has considerably hampered further studies of HHV-6-induced neuropathogenesis. In this study, we have characterized a new model for HHV-6 infection in mice expressing the human CD46 protein. Infection of CD46 transgenic mice with HHV-6A resulted in long-term persistence of viral DNA in the brains of infected animals and was followed by lymphocyte infiltration and upregulation of the CCL5 chemokine in the absence of clinical signs of disease. The secretion of a panel of chemokines was increased after infection in primary murine brain glial cultures, and the HHV-6-induced chemokine expression was inhibited when TLR9 signaling was blocked. These results describe the first murine model for HHV-6A-induced brain infection and suggest the importance of the TLR9 pathway in HHV-6A-initiated neuroinflammation.

Human herpesvirus (HHV-) 6A and HHV-6B are two distinctβ-herpesviruses which have been associated with various neurological diseases, including encephalitis, meningitis, epilepsy, and multiple sclerosis. Although the reactivation of both viruses is recognized as the cause of some neurological complications in conditions of immunosuppression, their involvement in neuroinflammatory diseases in immunocompetent people is still unclear, and the mechanisms involved have not been completely elucidated. Here, we review the available data providing evidence for the capacity of HHV-6A and -6B to infect the central nervous system and to induce proinflammatory responses by infected cells. We discuss the potential role of both viruses in neuroinflammatory pathologies and the mechanisms which could explain virus-induced neuropathogenesis.

Human herpesvirus (HHV)-6A and HHV-6B are two enveloped DNA viruses of β-herpesvirus family, infecting over 90% of the population and associated with several diseases, including exanthema subitum (for HHV-6B), multiple sclerosis and encephalitis, particularly in immunosuppressed patients. Animal models are highly important to better understand the pathogenesis of viral infections. Naturally developed neutralizing antibodies to HHV-6 or a related virus were found in different species of monkeys, suggesting their susceptibility to HHV-6 infection. Both HHV-6 DNA and infectious virus were detected in experimentally infected Cynomolgus and African green monkeys, although most animals remained clinically asymptomatic. Furthermore, HHV-6A infection was shown to accelerate the progression of AIDS (acquired immunodeficiency syndrome) in macaques and to lead to the development of neurological symptoms in the marmoset model. Humanized SCID (severe combined immunodeficiency) mice efficiently replicated HHV-6 and were also susceptible to coinfection with HHV-6 and HIV-1 (human immunodeficiency virus 1). As CD46 was identified as a receptor for HHV-6, transgenic mice expressing human CD46 may present a potentially interesting model for study certain aspects of HHV-6 infection and neuroinflammation.

Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCEAlphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly. V enezuelan equine encephalitis virus (VEEV) is a representative member of the New World (NW) alphaviruses, which circulate mostly in Central and South America (1). In nature, VEEV is transmitted by mosquito vectors between vertebrate hosts, in which it causes a highly debilitating disease, often resulting in meningoencephalitis and a frequently lethal outcome (2). Among humans, the mortality rates are below 1%, mostly among the elderly and the very young, but the disease often leads to neurological sequelae (3, 4). Due to its user-friendly characteristics, VEEV has the potential for development as a biological warfare agent. It can be propagated to very high infectious titers in many commonly used cell lines, is very stable in lyophilized form, and is exceptionally efficiently transmitted by aerosol. Therefore, some of the VEEV serotypes a...