Good news for Cell! Scientists have pinpointed the entry and exit ports of the new coronavirus inside our noses

In a study published Jan.
5 in the journal Cell, Dr.
Peter Jackson, a professor of pathology, microbiology and immunology at Stanford University, and his colleagues brought this possibility closer to reality
by pinpointing the pathways through which SARS-CoV-2, the virus of COVID-19, enters and leaves our nasal cells.
"Our upper respiratory tract is not only a launching pad for lung infections, but also for spreading to others
," Jackson said.

Jackson co-worked with Dr.
Raul Andino, a professor of microbiology and immunology at UCSF, on the study, which is the first to describe COVID-19 nasal infection
in molecular detail.
The study's lead authors are Chien-ting Wu, Ph.
D.
, and graduate student Ran Cheng, a former Stanford postdoctoral scholar, and Peter Lidsky, Ph.
D.
, and Yinghong Xiao, Ph.
D.
, postdoctoral scholars at
UCSF.

The nose and airway are made up of epithelial tissue consisting mainly of three types of cells: basal cells, goblet cells, and multi-rim cells, which make up about
80% of nasal epithelial cells.
Polycilia cells form a protective barrier that prevents viruses from entering the airways
.

Jackson and his colleagues amplified two structures found on multiciliated epithelial cells: cilia and microvilli
.
While both structures are well known, they have not previously been found to be related to
how viruses enter or leave cells within the airways.

Cilia are spaghetti-like appendages
where various cells grow to the outer surface.
A single nasal epithelial cell may have as many as 400 such whip-like chains on its surface facing the nasal cavity, all beating
continuously and harmoniously.
On top of them is a thin layer of protein called mucus — closely related to key proteins in mucus — on top of which is a layer of mucus
.

Jackson said mucin molecules can connect to each other to form a network structure similar to an elastic three-dimensional chain fence, preventing larger viruses such as SARS-CoV-2 from entering upper respiratory tract cells
.
The mucus envelops viral particles, bacteria, environmental debris, and cell-breaking garbage, and keeps the cells underneath moist
.

The upper respiratory tract epithelial cilia pass through this layer of mucus, and their synchronized pulsations create a wave that pushes the mucus and its encased particles, like a slow-flowing river, to a place
where it can be coughed up or swallowed and digested.

Another common feature of almost all animal cells is microvilli, small spines that extend from the surface of the cell, like little
fingers.
Microvilli can grab and transport subcellular particles and vesicles
.

To get a closer look at what happens in the early stages of viral infection, Jackson and his colleagues used a sophisticated tissue culture method to generate what they called airway epithelial organoids, which mimic normal airways
.
Although lacking vascular and immune cells, these organoids fully recapitulate the structure of nasal epithelial cells, including the intact mucus-mucus layer and well-developed poly-rim cells
.

The scientists inoculated
these cultures in the same Petri dish as SARS-CoV-2.
Through optical, electron microscopy, and immunochemical staining, they monitored viral entry, replication, and exit
from epithelial cells.

Only cilia cells are infected
.
Electron microscopy showed that the virus initially attached only to cilia
.
After 6 h incubation of the organoids with SARS-CoV-2, many viral particles spread from the tip down on both sides
of the cilia.
Even 24 h after inoculation, the virus replicates
in only a few cells.
Large-scale replication takes 48 hours
.

It also takes a full day or two for SARS-CoV-2 in real life to start replicating
in full.

By reducing levels of proteins in nasal epithelial cells that are essential for cilia formation, cilia are depleted, severely slowing SARS-CoV-2 infection
.

"It is clear that human cilia-nasal epithelial cells are the main site
of SARS-CoV-2 entry into nasal epithelial tissue," Jackson said.

The researchers suspected that the delay in infection was due to the virus having to pass through the airway mucus-mucin barrier, and they treated the airway organoids with a mucin-selective enzyme that breaks down the mucin network grid
.
Jackson said it accelerated the entry of the virus within 24 hours, from "almost undetectable" to "easily detectable," and he concluded that eliminating the mucin in this mesh could stop the mesh from preventing SARS-CoV-2 infection
in organoids.

In patients with rare diseases with primary ciliary dyskinesia, their ability to pulsate cilia is impaired or no longer synchronized, and mucus flow loses directionality
.

In the airway organoids produced by these patients, the virus attaches to cilia in a similar
way to normal cells.
After 24 h of seeding, cell infection rates were similar
to those of normal infected cells.
Normal-looking microvilli stand on the cell surface
.

But after 48 hours, the total number of cells infected by SARS-CoV-2 was much smaller — it could only infect surrounding cells — suggesting that once SARS-CoV-2 began replicating inside the infected cells, the virus relied on adequate mucus flow to help it spread
throughout the upper respiratory tract.

A May 2020 study in the journal Nature Communications, co-authored by Jackson, showed that ACE2 receptors are concentrated on
cilia in nasal epithelial cells.
The new cellular study shows that SARS-CoV-2 binds
to epithelial cilia through this receptor.

From there, Jackson says, the virus may slide across the mucus-mucus barrier in one of two ways: one is hopscotch-style jumps from the cilia side, jumping from one ACE2 molecule to the next until it reaches the body of the cell, where it fuses with the cell membrane and crawls in; Alternatively, by inserting cilia, take an internal freight elevator to reach the cell body
.

"Once the virus passes through this barrier," he says, "it can replicate
freely in the underlying cells.
" ”

The researchers also found that SARS-CoV-2, once inside the cell, induces the activity of enzymes within the cell, causing microvilli to enlarge and branch, like crazy cactus plants, until their tips stick out above
the mucus barrier.
Their number is increasing
.
24 hours after vaccination, many of the altered microvilli, often less than half the length of cilia, have turned into huge, branched, tree-like structures the size or larger of cilia, adorned with attached viral particles that can enter the mucus-mucus layer, where they can drift along rivers of mucus and infect other, more distant cells
.

The researchers pinpointed in the cells enzymes that were turned on in large numbers by SARS-CoV-2 infection, which led to the transformation
of microvilli.
Inhibiting these enzymes stops this mutation and greatly reduces the spread
of the virus to other cells.

Can a spray knock them all down?

Jackson and his colleagues hatched respiratory organoids with either of the other two respiratory viruses (the now-surging respiratory syncytial virus and the less common parainfluenza virus), as well as BA.
1 (a variant of the omicron strain) with similar results
.

Omicron is more contagious and, as expected, it infects respiratory organ multicilia cells
faster than older strains used for other SARS-CoV-2 experiments.
But inhibiting the virus's entry into or exit from airway cells has still proven effective, even against this highly infectious variant
.

Jackson said these viral entry mechanisms may be a common feature
of many respiratory viruses.

The discovery identifies a new target for a nasal drug that prevents unknown respiratory viruses, such as the one you encounter during a pandemic, from making their home
in your nose or throat by blocking cilia movement or microvilli hypertrophy.

Jackson said the substances used in these experiments may be optimized for nasal sprays shortly after exposure to respiratory viruses, or as prophylactic drugs
.

"Delaying the entry, exit, or spread of the virus with topical, short-term drugs will help our immune systems catch up and arrive in time, stop full-blown infections, and hopefully limit future pandemics
," he said.