An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly.

Eastern equine encephalitis virus (EEEV) is a representative member of the New World alphaviruses. It is pathogenic for a variety of vertebrate hosts, in which EEEV induces a highly debilitating disease, and the outcomes are frequently lethal. Despite a significant public health threat, the molecular mechanism of EEEV replication and interaction with hosts is poorly understood. Our previously published data and those of other teams have demonstrated that hypervariable domains (HVDs) of the alphavirus nsP3 protein interact with virus-specific host factors and play critical roles in assembly of viral replication complexes (vRCs). The most abundantly represented HVD-binding proteins are the FXR and G3BP family members. FXR proteins drive the assembly of vRCs of Venezuelan equine encephalitis virus (VEEV), and G3BPs were shown to function in vRC assembly in the replication of chikungunya and Sindbis viruses. Our new study demonstrates that EEEV exhibits a unique level of redundancy in the use of host factors in RNA replication. EEEV efficiently utilizes both the VEEV-specific FXR protein family and the Old World alphavirus-specific G3BP protein family. A lack of interaction with either FXRs or G3BPs does not affect vRC formation; however, removal of EEEV's ability to interact with both protein families has a deleterious effect on virus growth. Other identified EEEV nsP3 HVD-interacting host proteins are also capable of supporting EEEV replication, albeit with a dramatically lower efficiency. The ability to use a wide range of host factors with redundant functions in vRC assembly and function provides a plausible explanation for the efficient replication of EEEV and may contribute to its highly pathogenic phenotype.IMPORTANCE Eastern equine encephalitis virus (EEEV) is one of the most pathogenic New World alphaviruses. Despite the continuous public health threat, to date, the molecular mechanisms of its very efficient replication and high virulence are not sufficiently understood. The results of this new study demonstrate that North American EEEV exhibits a high level of redundancy in using host factors in replication complex assembly and virus replication. The hypervariable domain of the EEEV nsP3 protein interacts with all of the members of the FXR and G3BP protein families, and only a lack of interaction with both protein families strongly affects virus replication rates. Other identified HVD-binding factors are also involved in EEEV replication, but their roles are not as critical as those of FXRs and G3BPs. The new data present a plausible explanation for the exceptionally high replication rates of EEEV and suggest a new means of its attenuation and new targets for screening of antiviral drugs.

Hypervariable domain of nonstructural protein nsP3 of Venezuelan equine encephalitis virus determines cell-specific mode of virus replication.

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.

Pseudoinfectious Venezuelan equine encephalitis virus: a new means of alphavirus attenuation.

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.

Hypervariable domains of nsP3 proteins of New World and Old World alphaviruses mediate formation of distinct, virus-specific protein complexes.

Alphaviruses are a group of single-stranded RNA viruses with genomes of positive polarity. They are divided into two geographically isolated groups: the Old World and the New World alphaviruses. Despite their similar genome organizations and virion structures, they differ in many aspects of pathogenesis and interaction with the host cell. Here we present new data highlighting previously unknown differences between these two groups. We found that nsP3 proteins of Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV) form cytoplasmic complexes with different morphologies and protein compositions. Unlike the amorphous aggregates formed by SINV nsP3 and other Old World alphavirus-specific nsP3s, VEEV nsP3 forms unique, large spherical structures with striking symmetry. Moreover, VEEV nsP3 does not interact with proteins previously identified as major components of SINV nsP3 complexes, such as G3BP1 and G3BP2. Importantly, the morphology of the complexes and the specificity of the interaction with cellular proteins are largely determined by the hypervariable domain (HVD) of nsP3. Replacement of the VEEV nsP3 HVD with the corresponding domain of SINV nsP3 rendered this protein capable of interaction with G3BPs. Conversely, replacement of the SINV nsP3 HVD with that of VEEV abolished SINV nsP3's interaction with G3BPs. The replacement of natural HVDs with those from heterologous viruses did not abrogate virus replication, despite these fragments demonstrating very low levels of sequence identity. Our data suggest that in spite of the differences in morphology and composition of the SINV- and VEEV-specific nsP3 complexes, it is likely that they have similar functions in virus replication and modification of the cellular environment.

New PARP gene with an anti-alphavirus function.

Alphaviruses represent a highly important group of human and animal pathogens, which are transmitted by mosquito vectors between vertebrate hosts. The hallmark of alphavirus infection in vertebrates is the induction of a high-titer viremia, which is strongly dependent on the ability of the virus to interfere with host antiviral responses on both cellular and organismal levels. The identification of cellular factors, which are critical in orchestrating virus clearance without the development of cytopathic effect, may prove crucial in the design of new and highly effective antiviral treatments. To address this issue, we have developed a noncytopathic Venezuelan equine encephalitis virus (VEEV) mutant that can persistently replicate in cells defective in type I interferon (IFN) production or signaling but is cleared from IFN signaling-competent cells. Using this mutant, we analyzed (i) the spectrum of cellular genes activated by virus replication in the persistently infected cells and (ii) the spectrum of genes activated during noncytopathic virus clearance. By applying microarray-based technology and bioinformatic analysis, we identified a number of IFN-stimulated genes (ISGs) specifically activated during VEEV clearance. One of these gene products, the long isoform of PARP12 (PARP12L), demonstrated an inhibitory effect on the replication of VEEV, as well as other alphaviruses and several different types of other RNA viruses. Additionally, overexpression of two other members of the PARP gene superfamily was also shown to be capable of inhibiting VEEV replication.

Early events in alphavirus replication determine the outcome of infection.

Alphaviruses are a group of important human and animal pathogens. They efficiently replicate to high titers in vivo and in many commonly used cell lines of vertebrate origin. They have also evolved effective means of interfering with development of the innate immune response. Nevertheless, most of the alphaviruses are known to induce a type I interferon (IFN) response in vivo. The results of this study demonstrate that the first hours postinfection play a critical role in infection spread and development of the antiviral response. During this window, a balance is struck between virus replication and spread in vertebrate cells and IFN response development. The most important findings are as follows: (i) within the first 2 to 4 h postinfection, alphavirus-infected cells become unable to respond to IFN-ß, and this occurs before the virus-induced decrease in STAT1 phosphorylation in response to IFN treatment. (ii) Most importantly, very low, subprotective doses of IFN-ß, which do not induce the antiviral response in uninfected cells, have a very strong stimulatory effect on the cells' ability to express type I IFN and activate interferon-stimulated genes during subsequent infection with Sindbis virus (SINV). (iii) Small changes in SINV nsP2 protein affect its ability to inhibit cellular transcription and IFN release. Thus, the balance between type I IFN induction and the ability of the virus to develop further rounds of infection is determined in the first few hours of virus replication, when only low numbers of cells and infectious virus are involved.