Majority Of Virions Research Articles

“Self-inactivating” rabies viruses are susceptible to loss of their intended attenuating modification

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Ultrastructure and biological properties of Avian influenza viruses following cryopreservation

Ultrastructure and infectious activity of avian influenza virus (strain А/Chicken/Sivash/02/05 (H5N1)) following cryopreservation and low temperature storage at –20, –70, and –196°C during various terms from 25 days up to 143 months using electron microscopy, serological and virological methods were investigated. Avian influenza viruses strain А/Chicken/Sivash/02/05 (H5N1) is stored in the Collection of Pathogens of the National Scientific Center ‘Institute of Experimental and Clinical Veterinary Medicine’ (Kharkiv, Ukraine), which was granted the National Endowment of Ukraine status. The conducted study allowed to reveal on electronograms the ultrastructural changes in AIV during long term storage (18 months) at moderately low temperature (–20°C), in particular loss of glycoprotein of peplomers in the majority of virions. The changes in ultrastructure of the virus samples were accompanied by a loss of hemagglutinating activity during long-term storage of AIV samples at moderately low temperature of –20°C. When storing the AIV samples at –70 and –196°C the virions generally remain negatively contrasted, keep peplomers for the studied storage duration

First Insights into the Entry Process of Hyperthermophilic Archaeal Viruses

A decisive step in a virus infection cycle is the recognition of a specific receptor present on the host cell surface, subsequently leading to the delivery of the viral genome into the cell interior. Until now, the early stages of infection have not been thoroughly investigated for any virus infecting hyperthermophilic archaea. Here, we present the first study focusing on the primary interactions between the archaeal rod-shaped virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) (family Rudiviridae) and its hyperthermoacidophilic host, S. islandicus. We show that SIRV2 adsorption is very rapid, with the majority of virions being irreversibly bound to the host cell within 1 min. We utilized transmission electron microscopy and whole-cell electron cryotomography to demonstrate that SIRV2 virions specifically recognize the tips of pilus-like filaments, which are highly abundant on the host cell surface. Following the initial binding, the viral particles are found attached to the sides of the filaments, suggesting a movement along these appendages toward the cell surface. Finally, we also show that SIRV2 establishes superinfection exclusion, a phenomenon not previously described for archaeal viruses.

Cooperative Vaccinia Infection Demonstrated at the Single-Cell Level Using FluidFM

The mechanisms used by viruses to enter and replicate within host cells are subjects of intense investigation. These studies are ultimately aimed at development of new drugs that interfere with these processes. Virus entry and infection are generally monitored by dispensing bulk virus suspensions on layers of cells without accounting for the fate of each virion. Here, we take advantage of the recently developed FluidFM to deposit single vaccinia virions onto individual cells in a controlled manner. While the majority of virions were blocked prior to early gene expression, infection of individual cells increased in a nondeterministic fashion with respect to the number of viruses placed. Microscopic analyses of several stages of the virus lifecycle indicated that this was the result of cooperativity between virions during early stages of infection. These findings highlight the importance of performing controlled virus infection experiments at the single cell level.

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis.

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.

Directly Monitoring Individual Retrovirus Budding Events Using Atomic Force Microscopy

Retrovirus budding is a key step in the virus replication cycle. Nonetheless, very little is known about the underlying mechanism of budding, primarily due to technical limitations preventing visualization of bud formation in real time. Methods capable of monitoring budding dynamics suffer from insufficient resolution, whereas other methods, such as electron microscopy, do not have the ability to operate under physiological conditions. Here we applied atomic force microscopy to real-time visualization of individual Moloney murine leukemia virus budding events. By using a single-particle analysis approach, we were able to observe distinct patterns in budding that otherwise remain transparent. We find that bud formation follows at least two kinetically distinct pathways. The majority of virions (74%) are produced in a slow process (>45 min), and the remaining particles (26%) assemble via a fast process (<25 min). Interestingly, repetitive budding from the same site was seen to occur in only two locations. This finding challenges the hypothesis that viral budding occurs from distinct sites and suggests that budding is not restricted laterally. In this study, we established a method to monitor the fine dynamics of the virus budding process. Using this single-particle analysis to study mutated viruses will enable us to gain additional insight into the mechanisms of viral budding.

In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope.

Human antibody responses, or versions thereof, can be cloned as phage display libraries. In vaccine evaluation, the possibility therefore exists of challenging the human response in vitro, rather than in vivo, in order to assist in establishing the most promising vaccine leads. The characteristics of the antibodies retrieved directly indicate the strengths and weaknesses of the vaccine at the molecular level. We applied this approach to compare recombinant and native human immunodeficiency virus type 1 envelope preparations. We conclude that recombinant gp160, gp140, and, to a lesser extent, gp120 present epitopes around the CD4 binding site in a conformation different from that of the native multimer and contrary to expected vaccine requirements. Antibodies to the potently neutralizing b12 epitope were selected preferentially from an immune library by purified human immunodeficiency virus type 1 virions. This suggests that b12 is a major epitope on the virions, in contrast to recombinant envelope preparations, in which related, weakly neutralizing epitopes predominate. Although the majority of virions in the preparation used are expected to be noninfective, it appears that they predominantly express a native envelope configuration and would be able to elicit potent neutralizing antibodies.

Helical nucleocapsid structure of the oncogenic ribonucleic acid viruses (oncornaviruses).

Negative staining of virions and isolated nucleoids from avian myeloblastosis virus, murine leukemia virus, murine mammary tumor virus, and feline leukemia virus reveals common internal structures. The majority of virions that are penetrated by phosphotungstate show spherical nucleoids with no apparent symmetry. In a small percentage of virions, two distinctive structures are found: (i) single strands (3 to 5 nm in diameter) which are presumed to be the nucleoprotein and are found randomly oriented throughout the viral interior and (ii) helical structures (7 to 9 nm in diameter) which contain these nucleoprotein strands and are observed at the periphery of the nucleoid. The finding of helical nucleocapsid segments at the periphery of the nucleoid, as well as the hollow spherical structure observed in thin section of budding virions, has led to the hypothesis that the nucleocapsid of the freshly budded oncornavirus is supercoiled as a hollow sphere. This symmetry, however, is considered transient, as the internal structure of the extracellular virus undergoes a conformational rearrangement; thus, due to structural instability, the nucleocapsid uncoils and the nucleoprotein strands fill the interior of the virion. The extracellular virion is therefore considered degenerate in respect to symmetry, explaining the difficulty in detecting a helical nucleocapsid.