1. Freeze-Fracture of Electron Microscopy of HIV-1 p24 capsid protein interaction with the
Viral Envelope.
Eric R. Newman
Project Summary
The goal of this research is to further understand how HIV incorporates/attaches
the host cell's lipid membrane as a viral envelope to its RNA enveloping capsid. HIV and
other similar enveloped virus’s biggest competitive advantage is their ability to evade
the host's immune system by hiding behind host cell's membrane complete with host
cell recognition proteins. Research into how this viral envelope recognizes and attaches
to non-infected cells and the mechanism of budding through hijacking of the ESCRT
complex has been extensive. However, research into the actual attachment of the viral
envelope to the capsid is relatively new as it is not a conventional target for treatment.
The goal of this research is to visualize the underlying structure between the viral
envelope and capsid by freeze-fracture electron microscopy to further elucidate the
attachment of the viral envelope to the capsid. Of specific interest is the C-terminal
region of the CA capsid protein that has been shown in previous studies to bind to
lipids. Understanding of this binding event could lead to methods to disrupt this process,
thereby inhibiting infection by HIV and other enveloped viruses.
Specific Aims
The long term goal of this research is to further understand the mechanism of
HIV infection in human cells. Knowledge of how the viral envelope attaches/remains
secure to the capsid enables the possibility of utilizing this complex as a potential drug
or treatment target to prevent and inhibit HIV infection through stripping the virus of its
ability to hide from the host immune system. Without an attached viral envelope the HIV
capsids would be vulnerable to the traditional immune response pathways.
The specific aim of the proposed research is to directly visualize the attached
face of the HIV capsid and the corresponding bottom layer of the viral envelope for
qualitative and visual analysis of the connective elements between the two.
This aim will be carried out through arresting viral bud release by introducing a
premature stop codon into the Gag protein coding for the p6 domain of Pr55Gag,
Freeze-fracture coupled electron microscopy will then be employed to visualize the
arrested and forming viral buds in- and cross-plane with the fractured membrane for a
qualitative look at the capsid/envelope interaction.

2. Background and Significance
Human immunodeficiency virus (HIV) (Figure 1.) is an enveloped lentivirus
responsible for acquired immunodeficiency syndrome (AIDS). AIDS is a syndrome
designated by a low CD4+ T cell count and/or specific diseases and infections
correlated to HIV infection. The HIV virion is spherical with a ~120nm diameter, it is
composed of the viral envelope (phospholipid bi-layer) with its associated membrane
proteins (host and viral) and a protein capsid within which two copies of HIV ssRNA are
located. The capsid is conical and made up of an estimated 2000 copies of capsid
protein monomer. HIV is particularly difficult to treat due to its ability to infect non
dividing cells, genetic variability, and acquisition of host cell membrane during its
budding process. The characteristic of HIV as an enveloped virus is likely the largest
challenge faced by the host immune system in recognizing and eliminating it. As HIV
completes its replication cycle it forms a viral capsid containing two copies of the HIV
ssRNA genome (Moss, 2013). Through the production of proteins coded for by the pro-
viral genetic material integrated into the host cell genome and hijacking of native
proteins, this capsid begins to bud through the host cell membrane. The viral bud
releases from the host cell enveloped in the host’s cell membrane with its associated
proteins (Figure 1.). The integration of the hosts membrane bound proteins help the
virion avoid detection by the immune response. Before budding, bud sites are
proliferated with glycoprotein 41 (gp41) and glycoprotein 120 (gp120) proteins
responsible for the mature virions ability to bind and fuse to a host cell. HIV’s genetic
variability is a product of its rate of replication ~1010 virions daily, mutation rate of 3 x10-5
per nucleotide base pair per replication cycle and the genetic recombination ability
imparted to it through its reverse transcriptase-mediated into the host’s genetic material
(Moss, 2013). This
rate of mutation helps
keep the few elements
that might be
recognized by a host’s
immune system (e.g.,
gp120 and any
degraded protein
fragments picked up
by MHC class 1
molecules) too
variable to elicit any
effective immune
response. The same
challenges the
immune system faces
in targeting HIV are
faced by any
attempted medical
intervention. Though
there are conserved
regions within the
Figure 1. Mature HIV-1 Virion
Used without permission (http://www.scistyle.com/).

3. virus’s proteins and genome, much of it is variable and changes rapidly making a
vaccine unattainable so far, and anti-viral modification only moderately effective. Issues
such as these put constraints on what can be used as viable treatment targets and force
modern science to look at alternate non-conventional approaches to treat HIV.
Modern treatment of HIV consists of five antiviral drug classes. There are
currently 20 approved antiretroviral drugs with the majority being reverse-transcriptase
inhibitors and retroviral protease inhibitors (avert.org). The the most recent classes
consist of fusion of entry inhibitors and integrase inhibitors. The most recent two classes
have had the most success in maintaining virulency against HIV due to the conserved
nature of their targets, HIV's membrane bound glycoproteins and integrase. A issue
with current treatment is HIV's development of resistance to retroviral treatment leading
to a "first line" and "second line" strategy of therapy approach consisting of multiple
waves of a combination of drugs (avert.org).
Global funding in response to HIV/AIDS ranging from care & treatment through
prevention to research is estimated to be $31.7 Billion dollars for 2016 by The Henry J.
Kaiser Family Foundation (Figure 2.). The NIH reported funding for HIV/AIDS research
in the U.S. alone at $3 billion in 2014. According to the World Health Organization
(WHO) there are an estimate 35 million people currently infected by the HIV virus, with
2.1 million new infections during 2013, and HIV is the leading cause of infectious death
Figure 2. Proposed U.S. Federal funding request for HIV/Aids by category.

4. worldwide. Numbers
such as these
emphasize the demand
and importance in
furthering HIV research.
Synthesis of the
capsid protein is carried
out through a
polyprotein precursor the
group-specific antigen
protein (Gag) (Figure 3.)
that is cleaved by HIV
protease into capsid
protein, nucleocapsid,
mature matrix and Gag
proteins. Within the Gag protein
there is a proline rich 6-kDa domain
designated p6 located at the
proteins C-terminal (Figure 4.). This
p6 domain was shown to be integral
to viral bud release from the cell
membrane (Huang, Et al. 1995).
The p6 region consist of several
highly conserved domains implying
function. A nonsense mutation
consisting of a premature stop
codon at the very beginning of the
p6 domain reduced viral bud
release 20 fold (Huang, Et al. 1995).
The long term goal of the
proposed research is to exploit the
viral coat/capsid complex as a
treatment target. Recent
experiments have shown the C-
Terminal domain of the capsid
protein to have lipid binding
properties through fluorescence and
FTIR measurements and it is
believed that this is effect plays a
part in the conjoining of the capsid
and viral envelope (Figure
5.)(Barrera, Et al. 2006).
Understanding of capsid and viral
envelope functional morphology
through imaging of the capsid
Figure 3. Organization of the HIV-1 domains Capsid (CA),
Matrix (MA), Nucleocapsid (NC), protease p1 & p6 (PR),
integrase (IN) and RT domains of the gag and pol reading
frames. Used without permission (Huang, Et al. 1995).
Figure 4. Assembled HIV-1 Capsid composed of
~2000 monomeric copies (Left), Capsid protein
with N-terminal domain & C-Terminal domain of
interest (Top Right), Capsid Pentamer with NTD
displayed in blue (Middle Right), Capsid
Hexamer with NTD displayed in green (Bottom
Right). Used without permission (Deshmukh, Et
al. 2013).

5. protein's interaction with the fore mentioned envelope is intended to be a building block
to further more quantitative research into the strength and perturbations of this
attachment. The viral envelope is one of HIV’s greatest strengths allowing it to hide from
host immune response and an essential component in continuing the HIV life cycle. If
the viral coat could be disrupted or stripped from the HIV capsid it would not cure a
prevailing infection
but could be utilized
much as anti-virals
are today, as an
inhibitor to current
infections and
prevention against
possible infection by
at risk populations.
Disruption or removal
of the viral envelope
would shut down one
of HIV’s two
pathways to continue
its infection of new
cells and the only
pathway to infection
in a new host.
Without an intact viral
envelope the HIV
virion faces multiple
issues. First, without
the glycoproteins
located on and within
the viral envelope
HIV cannot recognize
and bind to the CD4
receptors and CCR5
& CXCR4 co-receptors on the host cell preventing fusion and infection with the inserted
viral genetic material (Figure 3.). Second, without the host proteins associated the
stolen lipid membrane the virion cannot effectively hide from the host’s immune
response leaving it vulnerable.
Freeze-etching is a subset of Freeze-fracturing that employs sublimation after
fracturing, both are techniques employed in tandem with electron microscopy. Electron
microscopy sample chambers are kept at low pressure to avoid electron scattering by
gasses which causes problems with biological samples. Freeze-fracturing freezes the
water within and around a biological sample at high pressure to from amorphous ice an
isochoric process. Fracturing of the frozen sample is conventionally done with a cold
razor blade. The fracture plane commonly splits the inner and outer lipid bilayer but can
also create a fracture plane splitting the lipid bilayer from the cell or split a plane directly
through a cell (Figure 6). When this is done transmembrane proteins are visible either
Figure 5. Crystal structure of monomeric Capsid protein C- terminal
domain with tyrosine & tryptophan residues in stick form. Regions
with predicted lipid membrane affinity are in dark gray including the
asterisk indicated alpha-helix. Used without permission from
(Barrera, Et al. 2006).

6. directly within a separated bi/monolayer or through the void they occupied. Once
sample fixation is achieved an additional step designated etching can be done where
more details of the fractured sample can be visualized if the surrounding ice is removed
through sublimation to maintain sample structure. This etching as done at low
temperature and high pressure to minimize ice crystal formation and remnant gas
deposition both of which contaminate the sample (Severs, 2007). Once the sample has
been prepared it is coated under an angle (~2-8o with rotation for small structure
resolution) with a thin film of heavy metal (generally platinum ~7nm) to create
shadowing for contrast and subsequently a layer of carbon (~15nm) at a 90o angle to
the fracture plane to stabilize the thin metal film. With a simultaneous evaporation of
platinum and carbon monoclonal steps of 11Å have been resolved (Bradley, 1959)
however more common platinum followed by carbon layering routinely have a resolution
of 2.5nm opposed to the 11Å resolution expected out of the methods described later.
Following sample shadowing and coating the remaining biological material is removed
using SDS or acid digestion after which the sample is ready for analysis (Servers,
2007).
Electron microscopy is a form of microscopy that uses an electron beam to
visualize a sample. Due to the extremely short wavelengths of electrons the resolving
power is much higher than that of conventional light microscopy. Transmission electron
Figure 6. (Top) Examples of different fracture planes obtained through Freeze-Fracture.
Exoplasmic Surface (ES), Plasmatic Fracture Face (PF), Exoplasmatic Fracture Face (EF),
Cytoplasm (Cyt). (Bottom) Freeze-Fracture with a PF and integrated proteins.
Used without permission (Source: http://www.leica-microsystems.com/science-lab/brief-
introduction-to-freeze-fracture-and-etching/)

7. microscopy (TEM) has a reported 50 pm resolution and can reach magnification up to
10,000,000x (Ernie, Et al. 2009). TEM utilizes an accelerated electron beam focused by
electromagnetic and electrostatic lenses into a focused beam that gets scattered as it
passes through the sample and the beam is subsequently used to expose a
photographic plate. (Ernie, Et al. 2009).
Altogether the proposed research is viable due to the following reasons. First,
bud arrestment fixing the subject to be studied and increasing the density per area of
buds approximately 20-fold will provide a large amount of sample to be studied (Huang,
et al. 1995). The mutagenesis to achieve this increased bud density has been
performed and verified by previous research (Huang, et al. 1995). Second, the C-
terminal region of the capsid protein has displayed lipid binding properties that imply
some sort of interaction with the lipid bi-layer viral envelope (Figure 5.)(Barrera, et al.
2006). Third, the long term goal of viral envelope degradation/removal would prevent
viral bud infection based on the gp120 protein function and viral envelope location and
gp40 location and function within the viral envelope, nonetheless the speculative
vulnerability of uncoated HIV virion to a host immune response (Figure 1.)(Maartens, et
al. 2014). Fourth, freeze-fracture has been demonstrated to provide fracture planes
through and around inter-membrane and membrane bound vesicles ~40 nm smaller
than the HIV virion (Zampighi, et al. 1999). Fifth, with freeze-fracture electron
microscopy's magnification and a resolving power of 11Å we expect to be able to
resolve the most likely culprit of capsid protein viral envelope interaction the C-terminal
alpha helix due to the 12Å diameter of alpha helices and the variable length a helix or
other subunit could protrude into the viral envelope. We hope to elucidate the
membrane bound and intermembrane proteins within, through and into the viral
envelope (Figure 5)(Ernie, et al. 2009). Last, there is a distinct need and available
funding for further information on HIV especially with pathways leading to conventional
and nonconventional treatment targets (Figure 2).
Research Design and Methods
First, arresting viral bud release will be achieved through site directed
mutagenesis followed by transfection. Viral bud visualization is challenging due to
multiple characteristics of the virion. The challenging characteristics of viral bud
visulization constituting of the viral bud size and migratory. Through site directed
mutagenesis a premature stop codon will be added right after the start codon of the p6
region of the HIV Gag protein (Figure 3.). The p6 region plays a crucial role in the
release of the HIV virion from the infected cell membrane once the viral capsid is fully
formed and disrupting it has been shown to arrest virion release from the host
membrane but still allow virion formation. Using a 1.2-kbp SphI-BalI fragment
(nucleotides 1442-2619) from a full length infectious clone of pNL4-3, the most widely
used vector for in vitro manipulation of the HIV proviral sequences. The 1.2-kbp SphI-
BalI fragment will then be sub-cloned into an M13mp18 single stranded DNA plasmid
and directed-oligonucleotide mutagenesis will be performed as proscribed by (Kunkle,
et. al., 1987). A 473-bp of ApaI-PpuMI fragment (nucleotides 2006-2483) will carry a
premature stop codon mutation in the (PTAP-, residues 7-10) region of the p6 domain of

8. the HIV Pr55Gag protein (Huang, et. al., 1995). This premature stop codon removing
the highly conserved PTAP- motif within the p6 region was shown to inhibit viral bud
release as designated in a 20 fold decrease in viral reverse transcriptase activity
(Huang. 1995). Arresting bud release will give a higher density of attached viral buds for
freeze fracture and subsequent electron microscopy imaging. Due to the small size of
retrovirus particles ~100nm and the inconsistent fracture planes of the freeze-fracture
technique an increased particle density will correlate to an increase in viral buds
fractured along a c-terminal plane while also being visualized in the subsequent electron
microscopy. Following the site directed mutagenesis the ApaI-PpuMI fragment will be
DNA sequenced as a positive control to verify the directed mutagenesis took place.
Two separate cell lines will be studied during the visualization the HIV capsid
stripped of either one or both of the lipid layers constituting its viral envelope in an
attempt to obtain different fracture planes during freeze-fracturing. Cell cultures
containing HeLa 10-15µm and CEM(12D-7) 5-10µm cells will be prepared and as
described in (Freed, et al., 1994). CEM(12d-7) cells are of the T-cell line native targets
for HIV. However, the HeLa cell line has been transfected to express Cluster
differentiation complex 4 (CD4) to allow infection by HIV. Calcium phosphate
precipitation and DEAE-dextran transfection will be used to transfect the HeLa and
CEM(12D-7) cells respectively, both methods described in (Freed, et al., 1994). The
CEM(12D-7) cell line will then be infected with equal amounts of mutated HIV after
normalization for reverse transcriptase activity as described by (Freed, Orenstein, et al.,
1994). Reverse Transcriptase assays for the CEM(12D-7) cell line will performed as
described by (Willey, et al., 1998) and (Freed, Orenstein, et al., 1994) for the HeLa cell
line as controls to determine the successful retardation of viral bud release by the
mutated HIV strain and HeLa cells. The afore mentioned normalization for RT activity is
executed to maintain a standard activity level for the subsequent RT assay to verify
virion release inhibition.
Forty-eight hours post infection, infected cells will be harvested and suspended in
distilled water to a resting culture at 4o-6o C with the sample being utilized before 12
hours has passed to minimize sample degradation. Obtaining and freezing of the HIV
sample by liquid nitrogen at -160oC will be executed as described by (Gross, Kuebler.,
1978). Following sample preparation, freeze-etching, platinum-carbon evaporation,
platinum-carbon shadow coating, and carbon backing will all be executed as described
in (Gross, Kuebler. 1978). Electron microscopy and image processing will also be done
using the procedure as designated by (Gross, Kuebler, 1978). Freeze fracturing will be
done on a modified high vacuum Balzers BA 350 U UHV-Freeze-Fracture apparatus
operating at ~10-9 torr and -196oC as opposed to the conventional ~10-6 torr and -100oC
for improved topographic resolution. The ultrahigh vacuum (UHV) technique was shown
to lessen structural distortion and contamination of the fracture plane with condensed
gasses hiding and create structures. Lowering the temperature during freeze-fracture
reduces distortion of fractured structures, damage caused during radiation heating and
surface diffusion by condensing metal atoms. The low vacuum helps alleviate the one
non-temperature dependent aspect of freeze fracturing, contamination of the sample by
gas molecules 1000-fold (Gross, Bas. 1978). After coating samples will be imaged with
a Siemens 102 electron microscope coupled with an anticontamination device with

9. pictures taken on electron image plates at all followed by detailed image analysis
encompassed in methods proscribed by (Gross, Kuebler., 1978).
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