You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the mechanism of entry. Discuss the important research questions you would ask and the types of experiments you might use to address the questions. You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the mechanism of entry. Discuss the important research questions you would ask and the types of experiments you might use to address the questions. Solution Answer: (There may be several questions related to the attachment and entry process, a few have been discussed) The first encounter between a virus and a target cell occurs at the plasma membrane. It is here that viruses first engage binding and entry receptors to initiate the infection process. Specific research questions on the mechanism of entry of this virus may be: 1. Adhesion and attachment: While normally non-specific in nature, different glycosphingolipids on the viral surface have been reported to confer cell specificity: the sialyllactose moiety on human immunodeficiency virus (HIV) membrane gangliosides, for instance, has been suggested to promote uptake of HIV into mature dendritic cells (mDCs). So, it can be asked whether the mechanism of entry is through specific glycosphingolipids and new roles of these glycosphingolipids? Experiments: Using liposomes to mimic the lipid composition and size of the virus, it can be demonstrate whether gangliosides are the key molecules that mediate liposome uptake. Liposome and VLP (virus like particle) capture assays on cultured cells, confocal microscopy and neuraminidase assay with VLP and LUV (large unilamellar vescicles) can be used for the investigation. 2. Lipid-mediated signalling for entry: Low-density lipoprotein receptors, negatively charged phospholipids and gangliosides have all been shown to assist entry of different viruses. Low-density lipoprotein receptors, also known as cholesterol receptors, are used by several members of the Flaviviridae family, including hepatitis C virus (HCV). The interaction is likely to occur through lipoproteins associated with the virion, such as cholesteryl esters and apolipoproteins (ApoB and ApoE) acquired during the assembly and budding of virus. So, it can be determined whether the LDL receptor acts as a receptor for this new virus and whether the endocytosis of the virus correlated with the level of LDL receptors?. Investigation of endocytosis of the virus by cultured cells in vitro can be demonstrated to have LDL receptors with the use of anti-LDL receptor antibody or DiI-LDL uptake. These cell lines can then be inoculated with a high titer virus positive serum. Intracellular viral RNA then can be detected by using ISH. To determine whether endocytosis of the virus correlated with the level of LDL receptor expression on cells, the well known modulatory effect of lipoproteins on the LDL receptor can be used to increase the number of LDL receptors (up-regulate) on cel.

1. You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the
mechanism of entry. Discuss the important research questions you would ask and the types of
experiments you might use to address the questions.
You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the
mechanism of entry. Discuss the important research questions you would ask and the types of
experiments you might use to address the questions.
Solution
Answer: (There may be several questions related to the attachment and entry process, a few have
been discussed)
The first encounter between a virus and a target cell occurs at the plasma membrane. It is here
that viruses first engage binding and entry receptors to initiate the infection process.
Specific research questions on the mechanism of entry of this virus may be:
1. Adhesion and attachment:
While normally non-specific in nature, different glycosphingolipids on the viral surface have
been reported to confer cell specificity: the sialyllactose moiety on human immunodeficiency
virus (HIV) membrane gangliosides, for instance, has been suggested to promote uptake of HIV
into mature dendritic cells (mDCs).
So, it can be asked whether the mechanism of entry is through specific glycosphingolipids and
new roles of these glycosphingolipids?
Experiments: Using liposomes to mimic the lipid composition and size of the virus, it can be
demonstrate whether gangliosides are the key molecules that mediate liposome uptake.
Liposome and VLP (virus like particle) capture assays on cultured cells, confocal microscopy
and neuraminidase assay with VLP and LUV (large unilamellar vescicles) can be used for the
investigation.
2. Lipid-mediated signalling for entry:
Low-density lipoprotein receptors, negatively charged phospholipids and gangliosides have all
been shown to assist entry of different viruses. Low-density lipoprotein receptors, also known as
cholesterol receptors, are used by several members of the Flaviviridae family, including hepatitis
C virus (HCV).
The interaction is likely to occur through lipoproteins associated with the virion, such as
cholesteryl esters and apolipoproteins (ApoB and ApoE) acquired during the assembly and
budding of virus.

2. So, it can be determined whether the LDL receptor acts as a receptor for this new virus and
whether the endocytosis of the virus correlated with the level of LDL receptors?.
Investigation of endocytosis of the virus by cultured cells in vitro can be demonstrated to have
LDL receptors with the use of anti-LDL receptor antibody or DiI-LDL uptake. These cell lines
can then be inoculated with a high titer virus positive serum. Intracellular viral RNA then can be
detected by using ISH.
To determine whether endocytosis of the virus correlated with the level of LDL receptor
expression on cells, the well known modulatory effect of lipoproteins on the LDL receptor can
be used to increase the number of LDL receptors (up-regulate) on cells by culturing in
lipoprotein-deficient media. Relative differences in endocytosis of LDL by various cultured cell
lines could be demonstrated by the specific uptake of DiI-LDL.
Experiments: LDL receptor assays and endocytosis assays.