Electron microscopy of frozen, hydrated lacrosse virus

1986 ◽  
Vol 44 ◽  
pp. 104-105
Author(s):  
Y. Talmon ◽  
B. V. V. Prasad ◽  
J. P. M. Clerx ◽  
W. Chiu ◽  
M. J. Hewlett
Keyword(s):  
Electron Microscopy ◽  
Ionic Strength ◽  
Nucleocapsid Protein ◽  
Rna Viruses ◽  
High Sensitivity ◽  
Bilayer Membrane ◽  
Viral Rna ◽  
California Serogroup ◽  
Viral Nucleocapsid ◽  
Negative Sense

LaCrosse (LAC) virus is a member of the California serogroup of the Bunyaviridae. The bunyaviruses are all negative-sense, RNA viruses whose genome consists of three segments of single stranded RNA. LAC virions are composed of multiple viral RNA strands complexed with viral nucleocapsid protein surrounded by a bilayer membrane embedded with 2 glycoproteins.Staining and drying of LAC virus samples produce images in which the virions are collapsed and distorted (Figure 1). Fixation prior to staining, also gives disappointing results, probably due to the high sensitivity of the LAC virus to changes in the ionic strength and composition of the solution in which it is suspended.

scholarly journals Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

2004 ◽  
Vol 78 (15) ◽  
pp. 8281-8288 ◽  
Author(s):  
M. A. Mir ◽  
A. T. Panganiban
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Viruses ◽  
Sin Nombre Virus ◽  
Viral Capsid ◽  
Genomic Rna ◽  
Rna Molecules ◽  
N Protein ◽  
High Affinity ◽  
Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

scholarly journals Reduced Nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition

2021 ◽  
Author(s):  
Benjamin E. Nilsson-Payant ◽  
Daniel Blanco-Melo ◽  
Skyler Uhl ◽  
Beatriz Escudero-Pérez ◽  
Silke Olschewski ◽  
...  
Keyword(s):  
Host Response ◽  
Measles Virus ◽  
Influenza A ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Machinery ◽  
Molecular Patterns ◽  
Negative Sense

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA, however its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data shows that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal amongst NSVs including Ebola virus, Lassa virus and Measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues towards developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines. IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus and Ebola virus. These viruses possess RNA genomes that are unreadable to the host as they require specific viral RNA dependent RNA polymerases in conjunction with other viral proteins such as nucleoprotein to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here we demonstrate the balance that negative-sense RNA viruses must achieve to both replicate efficiently and to avoid induction of the host defenses.

scholarly journals Evidence for gastrointestinal infection of SARS-CoV-2

2020 ◽  
Author(s):  
Fei Xiao ◽  
Meiwen Tang ◽  
Xiaobin Zheng ◽  
Chunna Li ◽  
Jianzhong He ◽  
...  
Keyword(s):  
Global Health ◽  
Respiratory Tract ◽  
Nucleocapsid Protein ◽  
Viral Rna ◽  
Immunofluorescent Staining ◽  
Transmission Route ◽  
Gastrointestinal Infection ◽  
Undetectable Level ◽  
Oral Transmission ◽  
Viral Nucleocapsid

AbstractThe new coronavirus (SARS-CoV-2) outbreak originating from Wuhan, China, poses a threat to global health. While it’s evident that the virus invades respiratory tract and transmits from human to human through airway, other viral tropisms and transmission routes remain unknown. We tested viral RNA in stool from 73 SARS-CoV-2-infected hospitalized patients using rRT-PCR. 53.42% of the patients tested positive in stool. 23.29% of the patients remained positive in feces even after the viral RNA decreased to undetectable level in respiratory tract. The viral RNA was also detected in gastrointestinal tissues. Furthermore, gastric, duodenal and rectal epithelia showed positive immunofluorescent staining of viral host receptor ACE2 and viral nucleocapsid protein in a case of SARS-CoV-2 infection. Our results provide evidence for gastrointestinal infection of SARS-CoV-2, highlighting its potential fecal-oral transmission route.

scholarly journals Two Novel Negative-Sense RNA Viruses Infecting Grapevine Are Members of a Newly Proposed Genus within the Family Phenuiviridae

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 685 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Beatriz Navarro ◽  
Francesco Di Serio ◽  
Kristian Stevens ◽  
Min Sook Hwang ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
High Throughput Sequencing ◽  
Movement Protein ◽  
Rna Viruses ◽  
Rt Pcr ◽  
Rna Dependent Rna Polymerase ◽  
Sequence Identity ◽  
The Family ◽  
Putative Movement Protein ◽  
Negative Sense

Two novel negative-stranded (ns)RNA viruses were identified by high throughput sequencing in grapevine. The genomes of both viruses, named grapevine Muscat rose virus (GMRV) and grapevine Garan dmak virus (GGDV), comprise three segments with each containing a unique gene. Based on sequence identity and presence of typical domains/motifs, the proteins encoded by the two viruses were predicted to be: RNA-dependent RNA polymerase (RdRp), nucleocapsid protein (NP), and putative movement protein (MP). These proteins showed the highest identities with orthologs in the recently discovered apple rubbery wood viruses 1 and 2, members of a tentative genus (Rubodvirus) within the family Phenuiviridae. The three segments of GMRV and GGDV share almost identical sequences at their 5′ and 3′ termini, which are also complementary to each other and may form a panhandle structure. Phylogenetics based on RdRp, NP and MP placed GMRV and GGDV in the same cluster with rubodviruses. Grapevine collections were screened for the presence of both novel viruses via RT-PCR, identifying infected plants. GMRV and GGDV were successfully graft-transmitted, thus, they are the first nsRNA viruses identified and transmitted in grapevine. Lastly, different evolutionary scenarios of nsRNA viruses are discussed.

scholarly journals Non-segmented negative-sense RNA viruses utilize N6-methyladenosine (m6A) as a common strategy to evade host innate immunity

2021 ◽  
Author(s):  
Mijia Lu ◽  
Miaoge Xue ◽  
Hai-Tao Wang ◽  
Elizabeth L. Kairis ◽  
Sadeem Ahmad ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Signaling Pathway ◽  
Type I Interferon ◽  
Rna Viruses ◽  
Viral Rna ◽  
Type I ◽  
Viral Rnas ◽  
Common Strategy ◽  
Host Innate Immunity ◽  
Negative Sense

N6-methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in non-segmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3)-knockout cells, we produced m6A-deficient virion RNA from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of type I interferon signaling pathway included by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity. IMPORTANCE The non-segmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There is no vaccine or antiviral drugs for many of these viruses. We found that representative members in the families of Pneumoviridae, Paramyxoviridae, and Rhabdoviridae in NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape the recognition by host innate immunity via a RIG-I dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon compared to m6A sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as a robust innate immunity will likely promote the subsequent adaptive immunity.

The ionic strength dependence of chromatin folding

1978 ◽  
Vol 36 (2) ◽  
pp. 220-221
Author(s):  
F. Thoma ◽  
TH. Koller
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Ionic Strength ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Carbon Supports ◽  
Ionic Strength Dependence ◽  
Chromatin Folding ◽  
Strength Dependence ◽  
Preparation Techniques

Under a variety of electron microscope specimen preparation techniques different forms of chromatin appearance can be distinguished: beads-on-a-string, a 100 Å nucleofilament, a 250 Å fiber and a compact 300 to 500 Å fiber.Using a standardized specimen preparation technique we wanted to find out whether there is any relation between these different forms of chromatin or not. We show that with increasing ionic strength a chromatin fiber consisting of a row of nucleo- somes progressively folds up into a solenoid-like structure with a diameter of about 300 Å.For the preparation of chromatin for electron microscopy the avoidance of stretching artifacts during adsorption to the carbon supports is of utmost importance. The samples are fixed with 0.1% glutaraldehyde at 4°C for at least 12 hrs. The material was usually examined between 24 and 48 hrs after the onset of fixation.

Application of Imaging Plate to High-Voltage Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 168-169
Author(s):  
Seiji Isoda ◽  
Kimitsugu Saitoh ◽  
Sakumi Moriguchi ◽  
Takashi Kobayashi
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
High Sensitivity ◽  
Imaging Plate ◽  
High Quality ◽  
Electron Dose ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Recording Material

On the observation of structures by high resolution electron microscopy, recording materials with high sensitivity and high quality is awaited, especially for the study of radiation sensitive specimens. Such recording material should be easily combined with the minimum dose system and cryoprotection method. Recently a new recording material, imaging plate, comes to be widely used in X-ray radiography and also in electron microscopy, because of its high sensitivity, high quality and the easiness in handling the images with a computer. The properties of the imaging plate in 100 to 400 kV electron microscopes were already discussed and the effectiveness was revealed.It is demanded to study the applicability of the imaging plate to high voltage electron microscopy. The quality of the imaging plate was investigated using an imaging plate system (JEOL EM-HSR100) equipped in a new Kyoto 1000kV electron microscope. In the system both the imaging plate and films can be introduced together into the camera chamber. Figure 1 shows the effect of accelerating voltage on read-out signal intensities from the imaging plate. The characteristic of commercially available imaging plates is unfortunately optimized for 100 to 200 keV electrons and then for 600 to 1000 keV electrons the signal is reduced. In the electron dose range of 10−13 to 10−10 C/cm2, the signal increases linearly with logarithm of electron dose in all acceralating volatges.

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