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The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses., Competing Interests: The authors have declared that no competing interests exist. Patent: 63/318008 Title: Humanized SARS-CoV-2 antibodies Co-inventors: A. Männik, KV, CE, BD, RLG, GK, EJ, PH, MUJ, MU, ES Patent: PCT/IB2021/059363 Title: SARS-Cov-2 neutralizing antibodies Co-inventors: GK, EJ, A.Männik, AP, DK, EŽ, MU, MUJ, (Copyright: © 2023 Hermet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Due to the high prevalence of infectious diseases and their concurrent outbreaks, there is a high interest in developing novel materials with antimicrobial properties. Antibacterial and antiviral properties of a range of metal-based nanoparticles (NPs) are a promising means to fight airborne diseases caused by viruses and bacteria. The aim of this study was to test antimicrobial metals and metal-based nanoparticles efficacy against three viruses, namely influenza A virus (H1N1; A/WSN/1933) and coronaviruses TGEV and SARS-CoV-2; and two bacteria, Escherichia coli and Staphylococcus aureus . The efficacy of ZnO, CuO, and Ag NPs and their respective metal salts, i.e., ZnSO 4 , CuSO 4 , and AgNO 3 , was evaluated in suspensions, and the compounds with the highest antiviral efficacy were chosen for incorporation into fibers of cellulose acetate (CA), using electrospinning to produce filter materials for face masks. Among the tested compounds, CuSO 4 demonstrated the highest efficacy against influenza A virus and SARS-CoV-2 (1 h IC50 1.395 mg/L and 0.45 mg/L, respectively), followed by Zn salt and Ag salt. Therefore, Cu compounds were selected for incorporation into CA fibers to produce antiviral and antibacterial filter materials for face masks. CA fibers comprising CuSO 4 decreased SARS-CoV-2 titer by 0.38 logarithms and influenza A virus titer by 1.08 logarithms after 5 min of contact; after 1 h of contact, SARS-COV-2 virus was completely inactivated. Developed CuO- and CuSO 4 -based filter materials also efficiently inactivated the bacteria Escherichia coli and Staphylococcus aureus . The metal NPs and respective metal salts were potent antibacterial and antiviral compounds that were successfully incorporated into the filter materials of face masks. New antibacterial and antiviral materials developed and characterized in this study are crucial in the context of the ongoing SARS-CoV-2 pandemic and beyond.

The high number of mutations in the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes its immune escape. We report a longitudinal analysis of 111 vaccinated individuals for their antibody levels up to 6 months after the third dose of the BNT162b2 vaccine. After the third dose, the antibody levels decline but less than after the second dose. The booster dose remarkably increases the serum ability to block wild-type or Omicron variant spike protein's receptor-binding domain (RBD) interaction with the angiotensin-converting enzyme 2 (ACE2) receptor, and these protective antibodies persist 3 months later. Three months after the booster dose, memory CD4 + and CD8 + T cells to the wild-type and Omicron variant are detectable in the majority of vaccinated individuals. Our data show that the third dose restores the high levels of blocking antibodies and enhances T cell responses to Omicron., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum., Competing Interests: The use of bovine colostrum as a prophylactic agent against SARS-CoV-2 has been patented (US patent application no 63/160,833) by Mario Plaas, K. Kogermann, E. Žusinaite, T. Tiirats, B. Aasmäe, A. Kavak, V. Poikalainen, L. Lepasalu, S. Piiskop, S. Rom, R. Oltjer, K. Kangro, E. Sankovski, J. M. Gerhold, R. Pert, A. Männik, A. Planken, A. Tover, M. Kurašin, M. Ustav and M. Ustav Jr.

Alphaviruses (family Togaviridae ) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.

Purpose: In Estonia, during the first wave of COVID-19 total number of cases confirmed by PCR was 13.3/10,000, similar in most regions, including capital Tallinn, but in the hotspot of Estonian epidemic, an island Saaremaa, the cumulative incidence was 166.1/10,000. We aimed to determine the prevalence of SARS-CoV-2 IgG antibodies in these two regions, symptoms associated with infection and factors associated with antibody concentrations., Methods: Participants were selected using stratified (formed by age decades) random sampling and recruited by general practitioners. IgG or neutralizing antibodies were determined from sera by four assays. Symptoms associated with seropositivity were analyzed by multiple correspondence analysis, antibody concentrations by multiple linear regression., Results: Total of 3608 individual were invited and 1960 recruited from May 8 to July 31, 2020. Seroprevalence was 1.5% (95% confidence interval (CI) 0.9-2.5) and 6.3% (95% CI 5.0-7.9), infection fatality rate 0.1% (95% CI 0.0-0.2) and 1.3% (95% CI 0.4-2.1) in Tallinn and Saaremaa, respectively. Of seropositive subjects 19.2% (14/73) had acute respiratory illness. Fever, diarrhea and the absence of cough and runny nose were associated with seropositivity in individuals aged 50 or more years. IgG, but not neutralizing antibodies concentrations were higher if fever, difficulty breathing, shortness of breath, chest pain or diarrhea was present, or hospitalization required., Conclusion: Similarly to other European countries the seroprevalence of SARS-CoV-2 in Estonia was low even in the hotspot region Saaremaa suggesting that majority of population is susceptible to SARS-CoV-2. Focusing only on respiratory symptoms may delay accurate diagnosis of SARS-CoV-2 infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The N6-methyladenosine (m 6 A) modifications in both viral and host cell RNAs play an important role in HIV-1 virus genome transcription and virus replication. We demonstrate here that activators of the METTL3/METTL14/WTAP RNA methyltransferase complex enhance the production of virus particles in cells harboring HIV-1 provirus. In parallel, the amount of m 6 A residues in the host cell mRNA was increased in the presence of these activator compounds. Importantly, the m 6 A methylation of the HIV-1 RNA was also enhanced significantly (about 18%). The increase of virus replication by the small-molecule activators of the METTL3/METTL14/WTAP complex excludes them as potential anti-HIV-1 drug candidates. However, the compounds may be of large interest as activators for the latent HIV-1 provirus copies deposited in host cells' genome and the subsequent virus eradication by an antiviral compound., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The Chikungunya virus (CHIKV) is an arbovirus belonging to the genus Alphavirus of the Togaviridae family. CHIKV is transmitted by the mosquitoes and causes Chikungunya fever. CHIKV outbreaks have occurred in Africa, Asia, Europe, and the countries of Indian and Pacific Oceans. In 2013, CHIKV cases were registered for the first time in the Americas on the Caribbean islands. There is currently no vaccine to prevent or medicines to treat CHIKV infection. The CHIKV nonstructural protease (nsP2) is a promising potential target for the development of drugs against CHIKV infection because this protein is one of the key components of the viral replication complex and is involved in multiple steps of virus infection. In this work, novel analogues of the potential CHIKV nsP2 protease inhibitor, first reported by Das et al. in 2016, were identified using molecular modeling methods, synthesized, and evaluated in vitro . The optimization of the structure of the inhibitor allowed to increase the antiviral activity of the compound 2-10 times. The possible mechanism of action of the identified potential inhibitors of the CHIKV nsP2 protease was studied in detail using molecular dynamics (MD) simulations. According to the MD results, the most probable mechanism of action is the blocking of conformational changes in the nsP2 protease required for substrate recognition and binding., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Chikungunya virus (CHIKV, family Togaviridae ) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1 to nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are the least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o'nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1, and FHL1 proteins were significantly reduced but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity. IMPORTANCE CHIKV outbreaks have affected millions of people, creating a need for the development of antiviral approaches. nsP3 is a component of the CHIKV RNA replicase and is involved in interactions with host proteins and signaling cascades. Phosphorylation of the HVD in nsP3 is important for the virulent alphavirus phenotype. Here, we demonstrate that nsP3 in CHIKV is phosphorylated and that the phosphorylation sites in the HVD are distributed in a unique pattern. Furthermore, the abrogation of some of the phosphorylation sites results in the attenuation of CHIKV, while abolishing all the phosphorylation sites completely blocked its replicase activity. Thus, the phosphorylation of nsP3 and/or the phosphorylation sites in nsP3 have a major impact on CHIKV infectivity. Therefore, they represent promising targets for antiviral compounds and CHIKV attenuation. In addition, this new information offers valuable insight into the vast network of virus-host interactions., (Copyright © 2021 American Society for Microbiology.)

Chikungunya fever results from an infection with Chikungunya virus (CHIKV, genus Alphavirus ) that is prevalent in tropical regions and is spreading fast to temperate climates with documented outbreaks in Europe and the Americas. Currently, there are no available vaccines or antiviral drugs for prevention or treatment of Chikungunya fever. The nonstructural proteins (nsPs) of CHIKV responsible for virus replication are promising targets for the development of new antivirals. This study was attempted to find out new potential inhibitors of CHIKV nsP2 protease using the ligand-based drug design. Two compounds 10 and 10c , identified by molecular docking, showed antiviral activity against CHIKV with IC 50 of 13.1 and 8.3 μM, respectively. Both compounds demonstrated the ability to inhibit the activity of nsP2 in a cell-free assay, and the impact of compound 10 on virus replication was confirmed by western blot. The molecular dynamics study of the interactions of compounds 10 and 10c with CHIKV nsP2 showed that a possible mechanism of action of these compounds is the blocking of the active site and the catalytic dyad of nsP2., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain-Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood-brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 10 4  plaque-forming units (p.f.u.) ml -1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Dengue virus (DENV) is associated with an estimated 390 million infections per year, occurring across approximately 100 countries in tropical and sub-tropical regions. To date, there are no antiviral drugs or specific therapies to treat DENV infection. Posaconazole and itraconazole are potent antifungal drugs that inhibit ergosterol biosynthesis in fungal cells, but also target a number of human proteins. Here, we show that itraconazole and posaconazole have antiviral activity against DENV. Posaconazole inhibited replication of multiple serotypes of DENV and the related flavivirus Zika virus, and reduced viral RNA replication, but not translation of the viral genome. We used a combination of knockdown and drug sensitization assays to define the molecular target of posaconazole that mediates its antiviral activity. We found that knockdown of oxysterol-binding protein (OSBP) inhibited DENV replication. Moreover, knockdown of OSBP, but not other known targets of posaconazole, enhanced the inhibitory effect of posaconazole. Our findings imply OSBP as a potential target for the development of antiviral compounds against DENV., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

Unlabelled: Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored., Importance: Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5-10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis.

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1-4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1-nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1-nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.