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Developing a VSV-G mediated entry assay for a mammalian fibroblast zoo

Shahraz Qamar – T-35 Summer Medical Intern

Langlois Lab

Department of Microbiology and Immunology

August 16, 2023

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Geoghegan and Holmes 2017

The Global Virome is massive

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Carlson et al Nature 2022

Climate change will dramatically impact virus transmission

The projected number of novel viral sharing events among mammal species in 2070

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Uncovering the rules for viral zoonosis

Modified from Plowright Nat Rev Micro 2017

Here are some of the barriers that exist that prevent viruses to jump to new species.
Human Exposure example
Generally speaking, its very hard for one virus to jump to another species. However, this does happen from time to time and when it does it can prove to be disastrous as we saw in the COVID-19 pandemic.
As we have seen in the last slide, climate change is forcing many different species to interact for the first time, and that creates an opportunity for viruses to be able to jump from one species to another.

Our goal is to see that when we remove some of the barriers and have a virus enter a cell of a species that it normally does not infect in the wild – can it replicate in that species?

So, if we take coronavirus for example. If before the pandemic, we had taken coronavirus from bats and infected human cells and we saw that it could replicate in human cells. Then we could reasonably assume that one of the barriers that is stopping the virus from infecting human cells is for example its ability to get in. And if it were to ever get in, maybe through a mutation that lets the spike protein interact with the ACE2 receptor then it would be likely that it would be able to cause significant infection in humans. Obviously, there are many more things that go into being able to infect a new species but those are some of the basics.

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Mammalian Fibroblast Zoo

Opossum (Monodelphis domestica)

Sugar glider (Petaurus breviceps)

Tenrec (Tenrec ecaudatus)

Armadillo (Dasypus novemcinctus)

Tree shrew

Human (Homo sapiens)

rhesus macaque (Macaca mulatta)

Ring tailed lemur

Marmoset

Rabbit (Oryctolagus cuniculus)

House mouse (Mus musculus)

Mouse (Mus spretus)

Syrian Hamster (Mesocricetus auratus)

North American deer mouse (Peromyscus maniculatus)

Oldfield mouse (Peromyscus polionotus)

California deer mouse (Peromyscus californicus)

White-footed deer mouse (Peromyscus leucopus)

Naked Mole Rat (Heterocephalus glaber)

Rat (Rattus norvegicus)

Thirteen-lined ground squirrel (Ictidomys tridecemlineatus)

Domestic Guinea Pig (Cavia porcellus)

Prairie vole (Microtus ochrogaster)

Meadow vole (Microtus pennsylvanicus)

Short-Tailed Shrew (Blarina brevicauda)

Domestic cat (Felus cattus)

Domestic Ferret (Mustela putorius furo)

Pig (Sus domesticus)

Sheep (Ovis aries)

Bat (Carollia perspicillata)

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“Fibroblast Zoo Project”

Measure viral replication

Tissue from available animals

Generate primary fibroblasts

Infect with virus

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Current Approach

Electroporating Cells

In vitro transcribed viral mRNA

qPCR to measure viral replication

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Hypothesis: VSV-G pseudotyped viruses bypass entry in all mammalian cells

Create a GFP expressing VSV-G pseudotyped lentiviral vector.
The VSV-G protein uses the Low-Density Lipoprotein Receptor (LDLR) to enter cells.
LDLR relatively well conserved in mammalian species.
Our hypothesis is that our GFP-expressing VSV-G pseudotyped lentiviral vector will be able to enter all our mammalian cells in our fibroblast zoo.
This would better mimic how viruses infect cells compared to the electroporation method.

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My Approach

Make viral particles

Transduce cells

Assess infectivity by fluorescence microscopy

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Making GFP expressing VSV-G Pseudotyped Lentivirus

Image from addgene

GFP

1. Experiment: VSV-G lentiviral GFP vector

2. Control: VSV-G (P127D mutant)

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Quantifying VSV-G psuedotyped infections in mammalian fibroblasts

Plate different cell types

A549 (Human)
MDCK (Dog)
CCL-141 (Duck)
DF-1 (Chicken)
NHDF (Human)
MEF (Mouse)

7. Vero (Monkey)

8. Cotton Rat

9. Raccoon

Plate cells

Infect

48 hours

Measure GFP

Cytation 5 machine

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Results

Immortalized cell lines

Bird cell lines

Primary mammalian fibroblasts

Here we see the results from our experiment.

So, we can see that we have 9 cell lines here and we can separate them out into 3 categories.

Number is our immortalized cell types

Then we have our bird species cells

lastly, we have our primary mammalian fibroblasts.

We can see in our immortalized cell types and the bird species cells we have very high levels of GFP signals. However, for our primary fibroblast we don't see high levels of GFP expression and that could be due to a number of factors. Generally speaking, primary fibroblast are harder to transduce compared to immortalized cell lines. Also, the primary fibroblast cells might be expressing higher quantity of antiviral restriction factors which makes it harder for them to get infected.

We also see that our mutant VSV-G (P127D) and our negative control show similar levels of GFP signals which is basically the background signal for our assay. This also confirms that our mutant VSV-G is a good negative control.

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Conclusions

VSV-G pseudotyped lentiviral GFP vector can infect some of our mammalian cell lines as seen with GFP expression.
VSV-G (P127D mutant) pseudotyped lentiviral vector is unable to infect our mammalian cells as there is no GFP expression.
Primary fibroblasts are relatively resistant to infection with our current VSV-G assay.

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Future Directions

Need to repeat these experiments and figure out if all of our mammalian fibroblasts can be infected with VSV-G.
Concentrating virus and infecting primary fibroblasts to see if we can get infection.
Might use a different virus (like influenza) instead of lentivirus to see if we can get infection.

Pseudotype the viruses that we currently have with VSV-G protein to

mimic a more natural viral infection in mammalian fibroblast cells

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The Matrix: To Do

~13 viruses
30 Species (in hand)
390 conditions

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Acknowledgements

Ryan Langlois

Clay Mickelson

Jordan Becker

Alison Barkhymer

William Matchett

Cera McDonald

Tristan Neal

Lauren Pross

Autumn Sanders

Dira Putri

Shanley Roach

Frances Shepherd

Esther Nyquist

Seyed Arad Moghadasi

Sofia Moraes

Adam Gilbertsen

Mescher Scholars Program

Dr. Daniel Mueller
Drew Keup

NIH T35 Medical Student Summer Research

Program in Infection and Immunity

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Questions?

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Extra Slides

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Results

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Results

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Results?