An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.
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ABSTRACT:
SUBMITTER: Monticelli SR
PROVIDER: S-EPMC8746760 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundLa Crosse virus (LACV), family Bunyaviridae, was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl with fatal encephalitis in La Crosse, Wisconsin. LACV is a major cause of pediatric encephalitis in North America and infects up to 300,000 persons each year of which 70-130 result in severe disease of the central nervous system (CNS). As an initial step in the establishment of useful animal models to support vaccine development, we examined LACV infectivity, pathogenesis, and immunogenicity in both weanling mice and rhesus monkeys.ResultsFollowing intraperitoneal inoculation of mice, LACV replicated in various organs before reaching the CNS where it replicates to high titer causing death from neurological disease. The peripheral site where LACV replicates to highest titer is the nasal turbinates, and, presumably, LACV can enter the CNS via the olfactory neurons from nasal olfactory epithelium. The mouse infectious dose50 and lethal dose50 was similar for LACV administered either intranasally or intraperitoneally. LACV was highly infectious for rhesus monkeys and infected 100% of the animals at 10 PFU. However, the infection was asymptomatic, and the monkeys developed a strong neutralizing antibody response.ConclusionIn mice, LACV likely gains access to the CNS via the blood stream or via olfactory neurons. The ability to efficiently infect mice intranasally raises the possibility that LACV might use this route to infect its natural hosts. Rhesus monkeys are susceptible to LACV infection and develop strong neutralizing antibody responses after inoculation with as little as 10 PFU. Mice and rhesus monkeys are useful animal models for LACV vaccine immunologic testing although the rhesus monkey model is not optimal.
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Project description:Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.
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Project description:We analyzed the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions and observed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only five miRNAs were found at a 2 to 4-fold higher level in virions than predicted based on random sampling. Of note, these included two miRNAs, miR-155 and miR-92a, reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding induces virion incorporation, we introduced artificial miRNA target sites into the HIV-1 genome and observed a 10 to 40-fold increase in the packaging of the cognate miRNA into virions, leading to the recruitment of up to 1.6 copies into each virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. We conclude that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to restrict cellular miRNA binding to their genome.
2016-12-22 | GSE89868 | GEO
Project description:Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.
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Project description:Viruses have developed many different strategies to counteract immune responses, and Vaccinia virus (VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here, we show that ISG15 participates in the control of VACV dissemination. Infection of Isg15-/- mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, compared to Isg15+/+ cells. Transmission electron microscopy revealed the accumulation of intracellular virus particles and a decrease in extracellular virus particles in the absence of interferon-stimulated gene 15 (ISG15), a finding consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Lastly, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, which are essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. IMPORTANCE Viral infections are a constant battle between the virus and the host. While the host's only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host's resources. Along millions of years of incessant encounters, poxviruses have developed a unique strategy consisting in the production two specialized "troops": intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination. The mechanism by which the virus "decides" to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.
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| S-EPMC3181475 | biostudies-literature
Project description:In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines.
| S-EPMC7111619 | biostudies-literature
Project description:Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome. The genomes of RNA viruses have the potential to interact with cellular miRNAs, which could lead to their incorporation into virions, with unknown effects on virion function. Here, it is demonstrated that wild-type HIV-1 virions essentially randomly incorporate low levels of the miRNAs expressed by infected cells. However, the specific incorporation of high levels of individual cellular miRNAs can be induced by insertion of cognate target sites into the viral genome. Of note, this results in a modest but significant inhibition of virion infectivity. These data imply that cellular miRNAs have the potential to inhibit viral replication by interfering with not only viral mRNA function but also virion infectivity.
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