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Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PV-infected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

Summary Zika virus (ZIKV) has been associated with morbidities such as Guillain‐Barré, infant microcephaly, and ocular disease. The spread of this positive‐sense, single‐stranded RNA virus and its growing public health threat underscore gaps in our understanding of basic ZIKV virology. To advance knowledge of the virus replication cycle within mammalian cells, we use serial section 3‐dimensional electron tomography to demonstrate the widespread remodelling of intracellular membranes upon infection with ZIKV. We report extensive structural rearrangements of the endoplasmic reticulum and reveal stages of the ZIKV viral replication cycle. Structures associated with RNA genome replication and virus assembly are observed integrated within the endoplasmic reticulum, and we show viruses in transit through the Golgi apparatus for viral maturation, and subsequent cellular egress. This study characterises in detail the 3‐dimensional ultrastructural organisation of the ZIKV replication cycle stages. Our results show close adherence of the ZIKV replication cycle to the existing flavivirus replication paradigm.

Plasma viremia reoccurs in most HIV-infected individuals once antiretroviral therapy is interrupted, and interindividual differences in the kinetics of viral rebound have been associated with virological and immunological factors. Antibody features, including Fc functionality and Fc glycosylation, have been identified as sensitive surrogates for disease activity in multiple diseases. Plasma viremia reoccurs in most HIV-infected individuals once antiretroviral therapy (ART) is interrupted. The kinetics of viral rebound, specifically the time until plasma virus becomes detectable, differ quite substantially between individuals, and associations with virological and immunological factors have been suggested. Standard clinical measures, like CD4 T-cell counts and plasma HIV RNA levels, however, are poor predictive markers. Antibody features, including Fc functionality and Fc glycosylation have been identified as sensitive surrogates for disease activity in multiple diseases. Here, we analyzed HIV-specific antibody quantities and qualitative differences like antibody-mediated functions, Fc gamma receptor (FcγR) binding, and IgG Fc glycosylation as well as cytokine profiles and cellular HIV DNA and RNA levels in 23 ART-suppressed individuals prior to undergoing an analytical ART interruption (ATI). We found that antibodies with distinct functional properties and Fc glycan signatures separated individuals into early and delayed viral rebounders (≤4 weeks versus >4 weeks) and tracked with levels of inflammatory cytokines and transcriptional activity of the viral reservoir. Specifically, individuals with early viral rebound exhibited higher levels of total HIV-specific IgGs carrying inflammatory Fc glycans, while delayed rebounders showed an enrichment of highly functional antibodies. Overall, only four features, including enhanced antibody-mediated NK cell activation in delayed rebounders, were necessary to discriminate the groups. These data suggest that antibody features can be used as sensitive indicators of HIV disease activity and could be included in future ATI studies. IMPORTANCE Plasma viremia reoccurs in most HIV-infected individuals once antiretroviral therapy is interrupted, and interindividual differences in the kinetics of viral rebound have been associated with virological and immunological factors. Antibody features, including Fc functionality and Fc glycosylation, have been identified as sensitive surrogates for disease activity in multiple diseases. Here, we systematically analyzed HIV-specific antibody quantities and qualitative differences in 23 ART-suppressed individuals prior to undergoing an analytical ART interruption (ATI). We found that antibodies with distinct functional properties and Fc glycan signatures separated individuals into early and delayed viral rebounders and tracked with levels of inflammatory cytokines and transcriptional activity of the viral reservoir. These data suggest that antibody features can be used as sensitive indicators of HIV disease activity and could be included in future HIV eradication studies.

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

Currently licensed influenza vaccines focus immune responses on viral hemagglutinin (HA), while the other major surface glycoprotein neuraminidase (NA) is not tightly controlled in inactivated vaccine formulations despite evidence that anti-NA antibodies reduce clinical disease. We utilized a bicistronic self-amplifying mRNA (sa-mRNA) platform encoding both HA and NA from four seasonal influenza strains, creating a quadrivalent influenza vaccine. sa-mRNA vaccines encoding an NA component induced the production of NA-inhibiting antibodies and CD4+ T-cell responses in both monovalent and quadrivalent formulations. Including NA in the vaccine enabled cross-neutralization against antigenically drifted strains and provided greater protection than HA alone upon A(H3N2) challenge in ferrets. These results demonstrate that next-generation bicistronic sa-mRNA vaccines expressing HA and NA induce potent antibodies against both viral coat proteins, as well as vaccine-specific cell-mediated immunity. When formulated as a quadrivalent seasonal influenza vaccine, the sa-mRNA platform provides an opportunity to increase the breadth of protection through cross-neutralizing anti-NA antibodies.

mRNA-based vaccines can be rapidly manufactured and have been demonstrated clinically to raise robust immune responses to COVID-19 and protect against severe COVID-19 disease. The clinical immunogenicity and efficacy of self-amplifying mRNA (sa-mRNA) vaccines have also been demonstrated, along with a longer duration of action than mRNA vaccines. However, a detailed understanding of differences between sa-mRNA and conventional mRNA vaccines with modified bases is lacking. Compared with a N1ψ-modified mRNA platform, when using an sa-mRNA approach, we observed a > 100-fold greater transfection efficiency for multiple antigens by sa-mRNA, all of which also showed high durability for gene-of-interest (GOI) production. The enhanced magnitude and durability of GOI expression by sa-mRNA compared with modified mRNA was also analysed in vivo using a luciferase reporter construct. In this experiment, sa-mRNA produced >100-fold cumulative bioluminescence compared with an mRNA construct. The elevation in GOI production translated into greater in vivo immunogenicity, where a 10-fold lower dose of sa-mRNA generated similar binding and neutralizing titers for the avian pandemic influenza H5N1 strain in both mouse and rat models. The sa-mRNA construct also generated comparable or higher antigen-specific CD8 T cell responses at 10-fold lower doses than mRNA. The lower doses of sa-mRNA generated a reduced elevation of reactogenic biomarkers while still generating similar or higher immunogenicity in rats and mice compared with modified mRNA. The current study suggests the potential of leveraging dose sparing, improved durability, enhanced immunogenicity, and possibly reduced reactogenicity of the sa-mRNA platform for vaccine applications. •GOI expression with sa-mRNA vaccine is more potent and durable vs. mRNA vaccine.•Antibody titers and CD8 T cell responses are greater with sa-mRNA vs. mRNA vaccines.•sa-mRNA vaccine generates similar immunogenicity as mRNA vaccine at smaller doses.•Dose-spared sa-mRNA vaccine induces lower cytokine release than mRNA vaccine.

Viruses are obligate intracellular parasites that replicate utilizing the resources of host cells. The replication of positive sense RNA viruses is coupled with alterations to host cell membranes. These viruses are believed to replicate efficiently by using co-opted membrane structures assembled from viral and host cell proteins and lipids. Poliovirus is a prototypical positive-sense RNA virus, however the topological details of viral RNA replication are not well understood. In this work we use electron cryotomography, among other methods, to examine the ultrastructure of fractionated poliovirus RNA replication factories that were formed within infected cells, and to investigate oligomeric interactions within a three dimensional crystal formed by a poliovirus polymerase point mutant. Investigation of the ultrastructure of isolated viral RNA replication factories shows that the low resolution features of cryopreserved membrane structures are essentially identical to previously observed structures within plastic sections of infected cells. Furthermore, greater detail visible using electron cryotomography reveals pore-like structures and other high energy membrane conformations within the replication factories. We see a mix of single, double, and multi-membrane structures that are arranged with openings that connect their interior lumenal space to the exterior environment. The lumen of some of these membranous structures contains a linear polymeric density thought to be RNA. We conclude that the RNA replication of poliovirus may occur on the lumenal surface of vesicular membranes with an opening to the cytoplasm for metabolite and product exchange. Within the poliovirus replication machinery, the principal component is the RNA polymerase 3Dpol. This prototypical RNA-dependent RNA-polymerase forms homo-oligomeric interactions that are key to its functions. To investigate these interactions, previous studies focused on hollow helical structures formed by wild-type polymerase. Here, we investigate the structure of small three-dimensional crystals formed by 3Dpol with a mutation of a single residue, lysine 314, to alanine. Using electron cryotomography and volume averaging, we demonstrate that the crystal packing within this point mutant does not include physiological polymerase-polymerase interactions. Elucidation of the topology of poliovirus replication machinery provides a basis for future development of antiviral therapeutics.

Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes.

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles secreted from host cells. Using biochemical and biophysical methods we identify multiple components in these secreted vesicles, including PV virions; positive and negative-sense viral RNA; essential viral replication proteins; ribosomal and regulatory cellular RNAs; and numerous host cell proteins, such as regulators of cellular metabolism and structural remodeling. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious extracellular vesicles containing both virions and a unique mat-like structure. Based on our biochemical data (western blot, RNA-Seq, and mass spectrometry), these mat-like structures are expected to be comprised of unencapsidated RNA and proteins. Our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral and host RNAs and proteins. The family of picornaviridae is comprised of small positive-sense RNA viruses, many of which are significant human pathogens. Picornaviruses exploit secreted extracellular vesicles for cell-to-cell viral transmission without cell lysis, and poliovirus serves as a model system for picornaviruses that are not protected by a surrounding membrane (non-enveloped viruses). The structure and contents of these vesicles secreted by virus-infected cells are described here. In addition to mature virions, these vesicles carry negative-sense, ‘template’ viral RNA and essential replication proteins, as well as cellular resources from the host. Their complex contents may comprise an enhanced virulence factor for propagation of infection, and understanding their structure and function is helping elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.