Download Chapter 5 Lodish 6E

Analyze the Data: Using RNAi
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by
short double-stranded RNA molecules called siRNA (small interfering RNAs). In
mammalian cells, transfection of 21–22 nucleotide siRNAs leads to degradation of
mRNA molecules that contain the same sequence as the siRNA. In the following
experiment, siRNA and knockout mice are used to investigate two related cell surface
proteins designated p24 and p25 that are suspected to be cellular receptors for the uptake
of a newly isolated virus.
a.
To test the efficacy of RNAi in cells, siRNAs specific to cell surface proteins p24
(siRNA–p24) and p25 (siRNA–p25) are transfected individually into cultured mouse
cells. RNA is extracted from these transfected cells and the mRNA for proteins p24 and
p25 are detected on Northern blots using labeled p24 cDNA or p25 cDNA as probes. The
control for this experiment is a mock transfection with no siRNA. What do you conclude
from this Northern blot about the specificity of the siRNAs for their target mRNAs?
b.
Next, the ability of siRNAs to inhibit viral replication is investigated. Cells are
transfected with siRNA–p24 or siRNA–p25 or with siRNA to an essential viral protein.
Twenty hours later, transfected cells are infected with the virus. After a further incubation
period, the cells are collected and lysed. The number of viruses produced by each culture
is shown below. The control is a mock transfection with no siRNA. What do you
conclude about the role of p24 and p25 in the uptake of the virus? Why might the siRNA
to the viral protein be more effective than siRNA to the receptors in reducing the number
of viruses?
Cell Treatment
Control
siRNA–p24
siRNA–p25
siRNA–p24 and siRNA–p25
siRNA to viral protein
Number of Viruses/ml
1  107
3  106
2  106
1  104
1  102
c.
To investigate the role of proteins p24 and p25 for viral replication in live mice,
transgenic mice that lack genes for p24 or p25 are generated. The loxP-Cre conditional
knock- out system is used to selectively delete the genes in cells of either the liver or the
lung. Wild type and knockout mice are infected with virus. After a 24-hour incubation
period, mice are killed and lung and liver tissues are removed and exam- ined for the
presence (infected) or absence (normal) of virus by immunohistochemistry. What do
these data indicate about the cellular requirements for viral infection in different tissues?
Tissue Examined
Mouse
Liver
Lung
Wild type
Knockout of p24 in liver
Knockout of p24 in lung
Knockout of p25 in liver
Knockout of p25 in lung
infected
normal
infected
infected
infected
infected
infected
infected
infected
normal
d.
By performing Northern blots on different tissues from wild-type mice, you find
that p24 is expressed in the liver but not in the lung, whereas p25 is expressed in the lung
but not the liver. Based on all the data you have collected, propose a model to explain
which protein(s) are involved in the virus entry into liver and lung cells. Would you
predict that the cultured mouse cells used in parts (a) and (b) express p24, p25, or both
proteins?
Solution
a.
The Northern blot reveals the steady state mRNA levels for p24 (in the center) and
p25 (on the right). For p24, only the transfection of siRNA-p24 reduces the amount
of p24 mRNA; whereas the control transfection without siRNA and transfection
with siRNA-p25 did not affect p24 mRNA levels. Similarly, for p25, only siRNAp25 reduced p25 RNA levels. These results demonstrate that the siRNAs can cause
specific degradation of their target RNAs.
b.
In the cultured cells, transfection of either siRNA-p24 or siRNA-p25 yielded a viral
titer that was slightly lower than the control transfection. This result indicates that
reduction of either p24 mRNA or p25 mRNA (and presumably the proteins encoded
by them) only minimally affects the ability of the virus to infect the cells. However,
transfection of both siRNA-p24 and siRNA-p25 did result in a significant reduction
in viral titer. In combination with the previous results, these results indicate that
either p24 or p25 can be used as a viral receptor. You could hypothesize that loss of
p24 and p25 mRNA would lead to a decrease in p24 and p25 protein, resulting in
inhibition of viral infection and replication. Transfection of siRNA to a viral
protein resulted in an even greater reduction of virus titer compared to transfection
with siRNA to both p24 and p25. One possible explanation is that transfection with
both either siRNA-p24 or siRNA-p25 leads to a reduction but not a complete
elimination of cell surface receptors for the virus, whereas siRNA to a viral protein
can inhibit viral replication/assembly directly. A second possibility has to do with
the half-life of p24 and p25 proteins. A reduction of p24 and p25 mRNA may not
lead to an immediate reduction in p24/p25 protein due to the stability of the protein.
The protein level is determined by the half-life of the protein and the length of time
after transfection of siRNA. For example, if the half lives of p24 and p25 proteins
are greater than 24 hours, then during the 20 hour incubation after transfection with
siRNA there should still be more than half of the protein still present. In this
problem no information about the half-life of the protein was given, so a definitive
conclusion cannot be made. But based on the results, it is reasonable to conclude
that the half-life of the p24 and p25 protein is relatively long, such that 20 hours
after transfection with the siRNA, sufficient protein remains to act as a receptor for
the virus.
c.
Using the loxP-Cre system, the p24 or p25 gene can be selectively knocked-out in
the liver or lung of transgenic mice. Using this approach, the receptor required for
viral infection in specific tissues can be examined in tissue sections by
immunohistochemistry. In the control, infection of a wild type mouse resulted in
viral infection in both liver and lung tissue. In the transgenic mice lacking the p24
gene in lung, viral infection was seen in both the liver and lung. These results
indicate that p24 protein is not required for viral infection of lung tissue. We cannot
determine from this one piece of data alone whether or not p24 is required in the
liver, as these mice still have a functional p24 gene in the liver. However, the mice
which have p24 knocked out in the liver had no viral infection in the liver,
indicating that p24 is required for viral infection in the liver. These mice show the
expected viral infection of the lung, because their lung tissue was not affected by the
selective knockout of p24 in the liver. Similarly, mice with a knockout of the p25
gene in liver displayed infection of the liver, demonstrating that p25 is not required
for viral infection in the liver. Mice with a knockout of the p25 gene in lung had no
viral infection in the lung, indicating that p25 is required for viral infection in lung.
These results are somewhat unexpected because the cell culture experiment in part b
indicated that either p24 or p25 could be used as a receptor for viral infection. But
these knockout experiments indicate that in liver tissue, removal of p24 function
alone is sufficient to prevent infection. Likewise, in lung tissue, removal of p25
function alone is sufficient to prevent infection.
d.
With the added information that liver tissue expresses only p24 and lung tissue
expresses only p25, the apparent discrepancy between the results from the knockout
mouse study and the cell culture studies can be resolved. Both the p24 and p25
protein can be used as a viral receptor in cells. But both proteins are not expressed
in liver tissue; only p24 protein is present. Because p25 is absent, p24 is the protein
used in liver as the viral receptor, and knockout of p24 in liver blocks viral
infection. Similarly, in the lung, p25 is expressed, but p24 is not. Thus in lung, p25
is the protein used as the viral receptor, and knockout of p25 in lung blocks viral
infection. The Northern blot in part a shows that in the cultured cells both p24 and
p25 are present. Thus in the cultured cells both p24 and p25 can act as the viral
receptor protein.