Mature Virus Particles Research Articles

Designing a recombinant coat protein to reduce tobacco mosaic virus infection in plants

The Tobacco Mosaic Virus (TMV) is a critical plant virus that can cause a significant drop in crop yield. To understand how recombinant coat-protein impacts the affinity and assembly of TMV’s subunits, research is being conducted to assess the effect of recombinant protein on virus resistance. To develop a recombinant coat-protein that can lower TMV infection rates in plants, a design strategy was employed that involves creating defective viral subunits leading to incorrect assembly. This method is similar to using defective puzzle pieces that form incorrect connections resulting in disrupted viral assembly, ultimately affecting the production of mature virus particles. The study investigated the effect of mutations on one side of the Tobacco mosaic virus coat-protein using molecular modeling and dynamics simulation techniques. The simulation showed that the recombinant subunit had lower flexibility (between 0.15 to 0.20 nm) compared to the other subunits (between 0.45 to 0.75 nm), which was attributed to the smaller loop area. The study suggests an effective recombinant coat-protein with the potential to prevent virus infection by disrupting the coat-protein assembly process. This approach can be used to design a plant vaccine against viruses. Developing a recombinant protein can also provide benefits to plants such as protection from pests and enhancement of growth and productivity.

Complex transcriptional regulations of a hyperparasitic quadripartite system in giant viruses infecting protists

Hyperparasitism is a common pattern in nature that is not limited to cellular organisms. Giant viruses infecting protists can be hyperparasitized by smaller ones named virophages. In addition, both may carry episomal DNA molecules known as transpovirons in their particles. They all share transcriptional regulatory elements that dictate the expression of their genes within viral factories built by giant viruses in the host cytoplasm. This suggests the existence of interactions between their respective transcriptional networks. Here we investigated Acanthamoeba castellanii cells infected by a giant virus (megavirus chilensis), and coinfected with a virophage (zamilon vitis) and/or a transpoviron (megavirus vitis transpoviron). Infectious cycles were monitored through time-course RNA sequencing to decipher the transcriptional program of each partner and its impact on the gene expression of the others. We found highly diverse transcriptional responses. While the giant virus drastically reshaped the host cell transcriptome, the transpoviron had no effect on the gene expression of any of the players. In contrast, the virophage strongly modified the giant virus gene expression, albeit transiently, without altering the protein composition of mature viral particles. The virophage also induced the overexpression of transpoviron genes, likely through the indirect upregulation of giant virus-encoded transcription factors. Together, these analyses document the intricated transcriptionally regulated networks taking place in the infected cell.

Rabies, The Administration of Vaccines and Public Health Concern, Neglected Challenges and Historical Perspective in China

Rabies virus is transmitted via contact of saliva of a rabid animal with a person ,s mucosa or a skin lesion. It is a fatal encephalomyelitis caused by members of expanding Lyssavirus genus, and the genus included 17 species. The aim of this literature review is to survey on rabies and vaccination in China, threats and challenges to eliminate it, especially in China. The information provided is obtained from randomized control experiments, review articles, and analytical observations and studies which were gathered from various literature sources such as Scopus, Google Scholar, PubMed, and Science Direct. The keywords were dogs, glycoprotein, lyssavirus, post-exposure prophylaxis, and rabies virus. Rabies virus is a bullet shaped enveloped virion, and the classical rabies virus and its field strains are discovered worldwide and induces to rabies in animals and humans. In recent years, China has made wonderful and significant achievements in rabies prevention and control, and currently, the mortality and incidence rate of human infection with rabies have decreased to the minimum level historically, which has caused a notable foundation for the ultimate elimination of human rabies. Generally, five proteins are available in mature rabies virus particles, phosphoprotein, nucleoprotein, glycoprotein, matrix protein, and RNA-dependent RNA polymerase. The main carriers of rabies in China are dogs which are accountable for most of the human rabies deaths in China. Most common way of entry of rabies virus into the body is both via infected neural tissue through open cuts in the skin and salvia. The most notable parameters which limit rabies vaccinations are limited laboratory-based surveillance, diminished community outreach and education, underestimation of the population at risk, irregular vaccine applications in space and time, inaccessible naïve free-ranging dogs, exclusion of puppies, cold chain failure, translocation of cases, effect of other species, including cats, wildlife, and livestock, accessibility to remote hotspots, competing priorities, lack of adequate resources, impact of poverty on responsible social ownership and routine animal health care, cultural disparities, Sisyphean fatigue, frustration and staff burn-out. Rabies remain a public health problem and continue to present health risks for both animals and human; It is important to improve access to PEP in endemic countries where human rabies deaths still happen, but the big problem is its costs that should be curtailed and surveillance strengthened for controlling and eliminating it.

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

Pr-independent biogenesis of infectious mature Zika virus particles.

Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.

Functions of the UL51 protein during the herpesvirus life cycle.

The herpesvirus UL51 protein is a multifunctional tegument protein involved in the regulation of multiple aspects of the viral life cycle. This article reviews the biological characteristics of the UL51 protein and its functions in herpesviruses, including participating in the maintenance of the viral assembly complex (cVAC) during viral assembly, affecting the production of mature viral particles and promoting primary and secondary envelopment, as well as its positive impact on viral cell-to-cell spread (CCS) through interactions with multiple viral proteins and its key role in the proliferation and pathogenicity of the virus in the later stage of infection. This paper discusses how the UL51 protein participates in the life cycle of herpesviruses and provides new ideas for further research on UL51 protein function.

Membrane curvature sensing and symmetry breaking of the M2 proton channel from Influenza A.

The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis

BackgroundEnterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive.MethodsA siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein’s GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A’s host interacting partners during infection.ResultsSmall GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A’s involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A’s top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection.ConclusionsThis study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Advances in the immunoescape mechanisms exploited by alphaherpesviruses.

Alphaherpesviruses, categorized as viruses with linear DNA composed of two complementary strands, can potentially to induce diseases in both humans and animals as pathogens. Mature viral particles comprise of a core, capsid, tegument, and envelope. While herpesvirus infection can elicit robust immune and inflammatory reactions in the host, its persistence stems from its prolonged interaction with the host, fostering a diverse array of immunoescape mechanisms. In recent years, significant advancements have been achieved in comprehending the immunoescape tactics employed by alphaherpesviruses, including pseudorabies virus (PRV), herpes simplex virus (HSV), varicella-zoster virus (VZV), feline herpesvirus (FeHV), equine herpesvirus (EHV), and caprine herpesvirus type I (CpHV-1). Researchers have unveiled the intricate adaptive mechanisms existing between viruses and their natural hosts. This review endeavors to illuminate the research advancements concerning the immunoescape mechanisms of alphaherpesviruses by delineating the pertinent proteins and genes involved in virus immunity. It aims to furnish valuable insights for further research on related mechanisms and vaccine development, ultimately contributing to virus control and containment efforts.

Morphogenesis of largemouth bass ranavirus (LMBRaV) in the epithelioma papulosum cyprinid cell line

Largemouth bass ranavirus (LMBRaV) belongs to the Ranavirus genus of the Iridoviridae family. It is a highly pathogenic virus that causes mass mortality in largemouth bass. In recent years, outbreaks of LMBRaV have been found in various provinces throughout China. Previous research mainly focused on virus isolation, identification, and detection, while the morphological change of the virus was still unknown. In this study, the ultrastructural morphogenesis of LMBRaV in epithelioma papulosum cyprinid (EPC) cells was observed and studied by using transmission electron microscopy. EPC cells were infected with LMBRaV (MOI=0.1) and then examined at 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h post infection. LMBRaV entered cells through endocytosis or direct penetration of cell membrane. After entering, the virus was observed in vesicles or lysosomes. After capsid uncoating, the virus genomes passed through the nuclear membrane and entered the cell nucleus. Virus genomes completed replication in the nucleus then transferred into the cytoplasm. In the cytoplasm, the progeny virus was assembled in the viromatrix and then aggregated in pseudocrystalline array. Finally, mature virus particles released through budding release from the cell membrane. Mature virus particles had a hexagonal shape and a diameter of approximately 150 nm. This study revealed the process of morphogenesis of LMBRaV in EPC cell line, providing essential information for further research on pathogenic mechanisms and immunological prevention of LMBRaV.

Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling.

Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2′ acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.

Single-Molecule Visualization of Bunyavirus Genome Segments Using Fluorescence In Situ Hybridization.

The genome of most bunyaviruses is divided over three (S, M, and L) single-stranded RNA segments of negative polarity. The three viral RNA segments are essential to establish a productive infection. RNA fluorescence in situ hybridization (FISH) enables the detection, localization, and quantification of RNA molecules at single-molecule resolution. This chapter describes an RNA FISH method to directly visualize individual segment-specific bunyavirus RNAs in fixed infected cells and in mature virus particles, using Rift Valley fever virus as an example. Imaging of bunyavirus RNA segments is a valuable experimental tool to investigate fundamental aspects of the bunyavirus life cycle, such as virus replication, genome packaging, and virion assembly, among others.

Efficient delivery of the immunodominant genes of African swine fever virus by adeno-associated virus serotype 2.

Adeno-associated virus serotype 2 (AAV2) represents a promising basis for developing a virus-vector vaccine against African swine fever (ASF). This study aimed to create genetic constructs based on AAV2 to deliver the immunodominant genes of ASF virus (ASFV) and to evaluate their functionality in vitro. The efficiency and specificity of transgene expression, as well as their non-toxicity in cells of target animals, were evaluated. Bioinformatics analysis methods were used to identify the immunodominant genes of ASFV. The target genes B646L, E183L, CP204L, and CP530R were identified and subsequently cloned into the pAAV-MCS vector. Assembly of recombinant AAV2 (rAAV2) was performed by cotransfection of AAV293 cells with the following plasmids: pAAV-MCS with the gene of interest, envelope, and packaging. Quantitative polymerase chain reaction was used to determine the AAV2 titer. The functionality of the constructs was evaluated in HEK293 and SPEV cells by determining the presence of mature proteins in the cell lysate and the expression levels of messenger RNA. The specificity of the target proteins in cell lysates was confirmed by Western blotting. The proposed AAV2 assembly protocol makes it possible to achieve a concentration of mature viral particles of at least 280 billion/mL of virus-containing material. The rAAV2 could effectively transduce host SPEV cells. The expression of both cistrons was detectable during the transduction of cells; therefore, the combined expression of immunogens in the cells of target animals should be possible using this method. This study demonstrated the potential of using genetic constructs based on AAV2 for the delivery of ASFV genes in vitro.

Stability of African swine fever virus genome under different environmental conditions.

African swine fever (ASF), a globally transmitted viral disease caused by ASF virus (ASFV), can severely damage the global trade economy. Laboratory diagnostic methods, including pathogen and serological detection techniques, are currently used to monitor and control ASF. Because the large double-stranded DNA genome of the mature virus particle is wrapped in a membrane, the stability of ASFV and its genome is maintained in most natural environments. This study aimed to investigate the stability of ASFV under different environmental conditions from both genomic and antibody perspectives, and to provide a theoretical basis for the prevention and elimination of ASFV. In this study, we used quantitative real-time polymerase chain reaction for pathogen assays and enzyme-linked immunosorbent assay for serological assays to examine the stability of the ASFV genome and antibody, respectively, under different environmental conditions. The stability of the ASFV genome and antibody under high-temperature conditions depended on the treatment time. In the pH test, the ASFV genome and antibody remained stable in both acidic and alkaline environments. Disinfection tests revealed that the ASFV genome and antibody were susceptible to standard disinfection methods. Collectively, the results demonstrated that the ASFV genome is highly stable in favorable environments but are also susceptible to standard disinfection methods. This study focuses on the stability of the ASFV genome under different conditions and provides various standard disinfection methods for the prevention and control of ASF.

Death from the Nile: Empirical formula, molar mass, biosynthesis reaction and Gibbs energy of biosynthesis of the West Nile virus

From July to October, West Nile virus is the leading cause of mosquito born disease in Europe and North America. This paper reports for the first time a chemical and thermodynamic analysis of the West Nile virus particles, genome and proteins, as well as interactions with its host organism. The empirical formula of mature West Nile virus particles was found through the atom counting method. Based on the empirical formula, biosynthesis reactions were formulated, which describe the formation of new virus live matter. Based on the biosynthesis reactions, Gibbs energy of biosynthesis was determined, which represents the physical driving force for the production of viral and host cell components. Gibbs energy of biosynthesis of the West Nile virus was found to be several times more negative than that of its host tissues. Due to the more negative Gibbs energy of biosynthesis, the West Nile virus components are produced much faster than those of its host cells. This allows the virus to hijack the host cell metabolism. Therefore, the virus-host interactions of the West Nile virus were explained through chemical and thermodynamic analysis.

Biological and Structural Analyses of New Potent Allosteric Inhibitors of HIV-1 Integrase.

HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range. Akin to other compounds of this class, the INLAIs predominantly inhibit the late stages of HIV-1 replication. A series of high-resolution crystal structures revealed how these small molecules engage the catalytic core and the C-terminal domains of HIV-1 IN. No antagonism was observed between our lead INLAI compound BDM-2 and a panel of 16 clinical antiretrovirals. Moreover, we show that compounds retained high antiviral activity against HIV-1 variants resistant to IN strand transfer inhibitors and other classes of antiretroviral drugs. The virologic profile of BDM-2 and the recently completed single ascending dose phase I trial (ClinicalTrials.gov identifier: NCT03634085) warrant further clinical investigation for use in combination with other antiretroviral drugs. Moreover, our results suggest routes for further improvement of this emerging drug class.

Ultrastructural analysis of monkeypox virus replication in Vero cells.

In early May 2022, the first worldwide monkeypox virus (MPXV) outbreak was reported, with different clinical aspects from previously studied human monkeypox infections. Despite monkeypox medical importance, much of its biological aspects remain to be further investigated. In the present work, we evaluated ultrastructural aspects of MPXVasynchronous infections in Vero cells by transmission electron microscopy (TEM). The viral strain was isolated from a male patient infected during the 2022 outbreak. TEM analysis showed: (i) adhered intracellular mature virus particles before entry of the host cell; (ii) a reorganization of the rough endoplasmic reticulum cisternae into the so-called "mini-nuclei"structure associated with genome replication; and (iii) noticeably different sites within the viral factory presenting granular or fibrillar aspects. We also observed viral crescents, different MPXVparticle morphotypes, and cellular alterations induced by infection, such as changes in the cytoskeleton structure and multimembrane vesicles abundance. Taken together, to the best of our knowledge, these results revealed for the first-time ultrastructural aspects of different steps of the MPXVcycle.

The structure and dynamics of a fully packaged RNA virus.

The vast majority of known viruses that infect humans have their genetic program written in molecules of RNA. Such RNA viruses are thought to attain their final, mature form via self-assembly of individual capsid proteins directed by specific interactions with the RNA genome. Despite tremendous amount of experimental information about the average structure of the protein capsids, much less is known about the structure and dynamics of the RNA genomes. Here, we characterized the structure, dynamics and ion atmosphere in a bacteriophage MS2 virus using all-atom molecular dynamics simulations. Starting from available experimental information, we built a computational model of a mature MS2 virion containing a 3569 nucleotide RNA genome enclosed inside a mature protein capsid that in turn consists of a single maturation protein embedded within an icosahedral lattice of the protein capsid. Our two 2.5-microsecond replica simulations of the complete virion differing by the initial configurations of the RNA genome revealed the stabilizing effect of the RNA—protein interactions on the structure of the mature capsid and on the conformation of the RNA genome. The virus particle as a whole was observed to undergo a breathing-like motion, whereas parts of the RNA genome not bound specifically to the protein capsid explored a range of structural transformations while retaining their secondary structure. A set of simulations performed upon removal of the RNA genome characterized the effect of the RNA on the internal pressure in the virus and on its ion atmosphere. Taken together, our work introduces a mature viral particle as a system of high dynamic complexity where repulsive RNA-RNA interactions compete with specific RNA-protein binding, with the ion atmosphere serving as intermolecular glue holding the assembly together.

Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds.

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.