(siRNA)-mediated knockdown of CDK2 and CDK7, two promi-nent members of the CDK family that are effectively inhibitedby Roscovitine and Olomoucine and expressed in the cytoplasmof CD4+T cells where HIV-1 reverse transcription occurs
They took it a step further. They used both pharmacological inhibition of CDK and preformed siRNA knockdowns of CDK genes in ex vivo lymphocytes.
independently of viral coreceptors
They are trying to demonstrate that fusion of the virus is not what is inhibited by the drugs, but rather the activity of a host factor such as CDKs.
VSV-G-pseudotypedHIV-1
Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. VSV has a higher tropic range that HIV-1.
R5-tropic
It uses the CCR5 coreceptor.
IL-2
Interleukin-2 (IL-2) is a 15-kDa cytokine predominantly secreted by activated T cells and represents a key player in the cell-mediated immune response in allograft rejection.
D3/CD8
Antigens unique to T lymphocytes.
ispecific antibodies
A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that can simultaneously bind to two different types of antigen.
ex vivo
Ex vivo means “outside of a living body.” In this type of experiment, the living tissues are not created artificially but directly taken from a living organism. The experiment is then immediately conducted in a laboratory environment, with minimal alteration of the organism’s natural conditions.
Cyclin-dependent kinases have a recognized role for supportingHIV-1 gene transcription from chromosomal DNA (Manceboet al., 1997
Is this role specific to the provirus genome or universal among host genes?
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA
You can tell that this is coming out of Harvard and MIT.
Thisvariation appears to be primarily related to the relative presenceor absence of specific host proteins that can modulate the effi-cacy of HIV-1 replication by inhibiting specific steps of the virallifecycle.
This is why (they think) elite controllers remain largely virus free for many years. It has yet to be determined what the influence is of host proteins which modulate HIV-1 replication on disease progression.
lymphocytes
Lymphocytes are white blood cells that are also one of the body's main types of immune cells. They are made in the bone marrow and found in the blood and lymph tissue.
‘elite controllers
A small subset of people living with HIV who are able to maintain suppressed viral loads for years without antiretroviral therapy (ART).
eceptive field properties
Used to categorize neurons, can be used to determine what stimuli the neuron responds to.
(sensory) or actions (motor)
Single unit electrophysiological recording allowed researchers to distinguish beteen motor and sensory neurons.
ive specific insight into the type of stimulus to which the neuron is “tuned” (i.e., the stimulus that elicits a maximal change in neuronal activity from a baseline state). Such tuning defines a neuron’s re-ceptive field—the region in sensory space (e.g., the body surface or a specialized structure such as the retina) within which a specific stimulus elicits action potential response (Figure 1.16)
What is the receptive field? How is single unit electrophysiological recording used?
synaptic potentials
ynaptic potential refers to the potential difference across the postsynaptic membrane that results from the action of neurotransmitters at a neuronal synapse. In other words, it is the “incoming” signal that a neuron receives. There are two forms of synaptic potential: excitatory and inhibitory.
eceptor potentials
transmembrane potential difference produced by activation of a sensory receptor. A receptor potential is often produced by sensory transduction. It is generally a depolarizing event resulting from inward current flow
Extracellular record-ing is particularly useful for detecting temporal patterns of action potential activity and relating those patterns to stimulation by other inputs
Extracellular recording purpose
Intracellular recording can detect the smaller, graded changes in electrical potential that trigger action potentials
Intraellular recording
electrophysiological recording
Measures electrical activity of neuron/nerve cell
and send axons peripherally that terminate in sen-sory endings in skeletal muscles. (The ganglia that serve this same function for much of the head and neck are called cra-nial nerve ganglia; see the Appendix.) The central axons of these sensory neurons enter the spinal cord, where they terminate on a variety of central neurons concerned with the regulation of muscle tone—most obviously on the motor neurons that determine the activity of the related muscles. The motor neurons in the circuits are the efferent neurons, one group projecting to the flexor muscles in the limb, and the other to extensor muscles. Spinal cord interneurons are the third element of the circuit. The interneurons receive synaptic contacts from sensory afferent neurons and make synapses on the efferent motor neurons that project to the flexor muscles; thus, they are capable of modulating the in-put–output linkage.
How the mytatic reflex circuit works
cra-nial nerve ganglia
idk if important probs not
. The afferent neurons that control the reflex are sensory neurons whose cell bodies lie in the dorsal root ganglia
contain cell bodies for sensory nerves that carry sensory information to the spinal cord.
dendrites, axon terminals, and glial cell processes that together constitute what is called neuropil (Greek pilos, “felt”; see Figure 1.3C). The neuropil constitutes the re-gions between nerve cell bodies where most synaptic con-nectivity occurs
Neuropil
Nissl method is one example; this technique stains the nucleolus and other structures (e.g., ribosomes) where DNA or RNA is found
Nissl method
inally, nucleic acid probes with complimentary sequences can detect mR-NAs that encode genes expressed in neurons or glia using a method called in situ hybridization (
In situ hybridization?
86 billion neu-rons and at least that many glia.
How many glia?
glial stem cells are also found throughout the adult brain. These cells retain the capacity to proliferate and generate additional precursors or differentiated glia, and in some cases neurons.
What is a glia stem cell?
Microglial
They are primarily scavenger cells that remove cel-lular debris from sites of injury or normal cell turnover. In addition, microglia, like their macrophage counterparts, secrete signaling molecules—particularly a wide range of cytokines that are also produced by cells of the immune system—that can modulate local inflammation and in-fluence whether other cells survive or die.
Schwann cells
Lay down myelin in PNS
Oligodendrocytes
Lay down myelin on axons in CNS
ynaptic vesicle
What releases the NTs
three types of differentiated glial cells
KNOW
functions that are well established include maintaining the ionic milieu of nerve cells; modulating the rate of nerve signal propagation; modulating synaptic action by controlling the uptake and metabolism of neurotransmit-ters at or near the synaptic cleft; providing a scaffold for some aspects of neural development; aiding (or in some instances impeding) recovery from neural injury; providing an inter-face between the brain and the immune system; and facilitat-ing the convective flow of interstitial fluid through the brain during sleep, a process that washes out metabolic waste
Why are glias important?
Glia do not participate directly in synaptic transmission or in electrical signaling
What is a glia?
postsynaptic specializations are typically chemical synapses
Typical to synaptic transmission, but electrical synapses at gap junctions also occur in decreased prevalence .
(diagram from class is a synapse not a gap junction)
The process by which the information encoded by action potentials is passed on at synaptic contacts to a target cell is called synaptic transmission,
Synaptic transmission-- passing along an action potential at synapse
Relatively short axons are a feature of local circuit neurons, or interneurons, throughout the nervous
Interneurons-- short neurons, low distance (local transmission)
Projection neurons-- long distance
pre- and postsynaptic elements called the synaptic cleft
Synaptic cleft?
The number of inputs to a single neuron reflects the degree of conver-gence, while the number of targets innervated by any one neuron represents its divergence
Convergence vs divergence
The most obvious morphological sign of neuronal specialization for communication is the ex-tensive branching of neurons. The two most salient aspects of this branching for typical nerve cells are the presence of an axon and the elaborate arborization of dendrites that arise from the neuronal cell body in the form of dendritic branches (or dendritic processes; see Figure 1.3E). Most neu-rons have only one axon that extends for a relatively long distance from the location of the cell body. Axons may have branches, but in general they are not as elaborate as those made by dendrites. Dendrites are the primary targets for synaptic input from the axon terminals of other neurons and are distinguished by their high content of ribosomes, as well as by specific cytoskeletal proteins.
Axon and dendrites explained
protein-synthetic organelles such as the endoplasmic reticulum are largely excluded from axons and dendrites
What is a dendrite? a short branched extension of a nerve cell, along which impulses received from other cells at synapses are transmitted to the cell body.
Is the ER in axon and dendrite?
Mito-chondria, for example, tend to be concentrated at synapses in neurons,
Mitochondria concentrated where?
erve cells, or neurons, and supporting glial cells (also called neuroglia, or simply glia)
nerve cells, or neurons, and supporting glial cells (also called neuroglia, or simply glia)
electron microscopic studies
How gap junctions were first identified, proving Golgi kinda right.
. Sherrington, who had been working on the apparent transfer of electrical signals via reflex path-ways, called these specialized contacts synapses
Synapses: Sherrington
microscopic examination of nervous tissue stained with silver salts according to Golgi’s pioneering method, Cajal argued persuasively that nerve cells are discrete entities,
Neuron staining
Some biol-ogists of that era even concluded that each nerve cell was connected to its neighbors by protoplasmic links, forming a continuous directly interconnected nerve cell network, or reticulum (Latin, “net”). The Italian pathologist Camillo Golgi articulated and championed this “reticular theory” of nerve cell communication
Dumbass
plice variants
Allows multiple proteins to be encoded by same gene.
dISCuSSIonIn
Overall summation of the paper and it's key results:
Orgando et. al’s study SARS-CoV-2 Replication in Vero E6 Cells drew important conclusions regarding the similarities and differences between SARS-CoV and SARS-CoV-2. SARS-CoV and SARS-CoV-2 have an 80% genetic identity and both bind to the ACE2 receptor in host cells. They share highly conserved amino acid sequences and similar protein structures such as the spike protein which serve as potential drug targets. For this reason, SARS-CoV research can, in many cases, be translated to SARS-CoV-2 However, this study found many important differences between the two viruses. For example, upon each cell passaging it was discovered that SARS-CoV-2 rapidly acquired mutations in the S1 and S2 domains of the spike protein likely due to selective pressure. Common mutations included the substitution of Arg692 with Gln near the furin like cleavage site and a ten amino acid deletion which erases the cleavage site. These mutations were linked to a large plaque phenotype. It was also found that SARS-CoV-2 produced far more intracellular RNA than SARS-CoV, but unexpectedly produced 50 fold less infectious particles. This is most likely due to the fact that SARS-CoV-2 does not efficiently add spiked proteins to it’s progeny, resulting in inactive particles. Immunofluorescence microscopy revealed that antisera was strongly cross reactive between SARS-CoV and SARS-CoV-2 for several different drug target proteins, implying similar structure and reducing the need to identify separate antibodies for immunofluorescence or immunologic assays for each virus. Electron microscopy (EM) allowed the researchers to visualize that ultrastructural changes inflicted on host cells were similar between SARS-CoV and SARS-CoV-2. EM also revealed the differences and similarities between the viral infection cycle. It was established that both viruses were similarly sensitive to remdesivir, alisporivir, and hydroxychloroquine, but that SARS-CoV-2 was highly sensitive to pretreatment with prelygated interferon alpha, which completely inhibited the replication of the virus. This study shows that there is great similarity between the two viruses which can be put to good use through translation of research, but that there are some key differences to take into account.
RESuLTSRapid
Results important points:
Mutations in the furin like cleavage site were linked to a large plaque phenotype. Upon cell passaging, the SARS-CoV-2 S protein rapidly evolves, likely due to selection pressure and adaptation to host cells.
Viral intracellular RNA levels were much higher for SARS-CoV-2 than SARS-CoV, even though SARS-CoV-2 progeny levels were found to be 50 fold lower. This is likely due to SARS-CoV-2 inneficient addition of S proteins to virus particles.
SARS-CoV antibodies strongly cross reacted with SARS-CoV-2 targets.
Both viruses were almost equally effected by the viral inhibitors remdezivir, alisporivir, and chloroquin. However, SARS-CoV-2 was completely inhibited by pretreatment of cells with PFG-IFN-alpha.
The two viruses were observed to cause highly similar ultrastructural changes to the cell.
METHodSCell
Methods important points:
Intracellular RNA synthesis was analyzed by lysing cells, running intracellular RNA on a gel and then flourescently labeling viral RNA segments. Viral proteins were analyzed by lysing cells and preforming a Western Blott.
In order to observe S protein evolution, a cDNA library was generated and Next Generation Sequencing was preformed. A plaque picking experiment was also preformed in order to observe the effect of S protein evolution on plaque phenotype.
Drug target proteins for SARS-CoV and SARS-CoV-2 were observed by immunoflourescence, which revealed cross reactivity in antisera.
InTRoduCTIonFor
Important points from the introduction:
SARS-CoV-2 is a zoontic born virus.
There are seven different CoVs which infect humans.
The beta CoVs, namely SARS-CoV, SARS-CoV-2 and MERS-CoV, are zoontic and have caused outbreaks. The other human infecting CoVs, called endemic human CoVs, cause mild cold like sumptoms.
Most CoVs likely have their evolutionary origins in bats.
CoVs posses a large +ssRNA genome, 2/3rds of which is comprised of PP1a and PP1b, which encodes replicase polyproteins (RDRPs) which are cleaved into 16 cleavage products by Mprotease and PLprotease.
The proteins nsp3, nsp4, and nsp6 create a replication organelle (RO) by hijacking an intracellular membrane for both replicating the genome and producing virus mRNAs for structural proteins. These structural proteins include S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins.
Drug targets for the virus include the S protein, RdRp, RNA helicase, and viral methyl transferases.
SARS-CoV and SARS-CoV-2 share an 80% genetic identity and highly conserved protein structures. Both bind to the ACE2 receptor which is found on the surface of Vero E6 Cells. For these reasons, research on SARS-CoV can, in some cases, be translated to SARS-CoV-2.
cleavage site
Protein regions which are erased by site specific proteases.
furin
Enzyme which excises protein regions resulting in protein activation.
ultrastructural
Structures of a cell which can only be seen using an electron microscope. Ultra structural changes can be injurious to a cell.
cross- reactivity
When an antibody specific to an antigen has a competitive affinity for another antigen.
antisera
Blood serum with antibodies raised against specific antigens.
Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS- CoV-2 infection was substantially more sensitive to pre- treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that – upon passaging in Vero E6 cells – SARS- CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin- like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays
Summation of abstract: The authors researched the cytopathology, kinetics, and evolution of both SARS-CoV and SARS-CoV-2 in the Vero E6 cell line.
SARS-CoV-2 generated higher levels of intracellular RNA than SARS-CoV, even though it produced 50 fold less infectious particles.
Immunoflourescence microscopy showed that antisera against NSPs, membrane and nucleocapsid proteins were cross reactive between the two viruses due to highly conserved protein structure.
Electron microscopy revealed that ultrastructural changes caused by both viruses were similar. The mechanisms of cell injury were highly conserved between the viruses.
Both viruses experienced similar suppression when treated with inhibitors, but SARS-CoV-2 showed great sensitivity to pretreatment of cells with prelygated interferon alpha.
When cell passaging is preformed, SARS-CoV-2 acquires mutations in the furin-like cleavage site region of the S protein due to selective pressure. These mutations result in a larger plaque assay phenotype.
cytopathology
Diagnosis on a cellular level, usually based on tissues.