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MQP-BIO-DSA-3389

Macrophage Tropism and Antibody Neutralization Resistance of HIV-1


is Conferred by the Envelope Protein gp120


A Major QualiIying Project Report
Submitted to the Faculty oI the
WORCESTER POLYTECHNIC INSTITUTE
in partial IulIillment oI the requirements Ior the
Degree oI Bachelor oI Science
in
Biology and Biotechnology

by


Bryan Egge


February 25, 2008


APPROVED:


Paul Clapham, Ph.D. David S. Adams, Ph.D.
Program in Molecular Medicine Biology and Biotechnology
UMass Medical Center WPI Project Advisor
Major Advisor
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ABSTRACT


Mutations in surIace proteins gp120 and gp41 oI the human immunodeIiciency
virus-1 (HIV-1) have previously been shown to aIIect the aIIinity (tropism) oI the virus
Ior various host cells. Understanding this process could lead to methods to block viral
entry or to prevent switches in tropism. The goal oI this MQP was Iirst to conIirm the
tropism and neutralizing sensitivities oI three HIV-1 strains (Q43-378.2, Q43-378.c, and
SQ43-380.1) isolated Irom a single patient, as previously determined in our laboratory
(Peters et al., 2006). Then, various viral chimeras were created Irom the gp120 and gp41
proteins oI the three original viruses to determine the eIIects oI the gp120 and/or gp41
proteins on viral tropism and neutralization sensitivity. Our hypothesis was that
neutralizing antibodies in vivo may select Ior envelope sequences that protect nearby
critical envelope sites (around the receptor regions) Irom neutralization. However, these
antibody-resistant envelopes may be compromised Ior their capacity to interact with
CD4. Hence, as neutralizing antibodies Iorm in vivo, this may help induce a shiIt in
tropism. The data indicate that the gp120 alone is able to conIer macrophage tropism,
and that gp41 has no noticeable eIIect on the tropism oI the virus. Similarly, the data
indicate that gp41 has no visible eIIect on the resistance to a b12 antibody that reacts with
the CD4-binding domain on gp-120, and that gp120 sequences are the most likely cause
oI neutralization resistance.
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TABLE OF CONTENTS


TITLE PAGE ................................................................................................................................................1
ABSTRACT...................................................................................................................................................2
TABLE OF CONTENTS..............................................................................................................................3
ACKNOWLEDGEMENTS..........................................................................................................................4
BACKGROUND ...........................................................................................................................................5
HIV VIRUS OVERVIEW..............................................................................................................................5
GENETICS OF HIV INFECTION..................................................................................................................6
HIV ENVELOPE STRUCTURE AND FUNCTION...........................................................................................7
HIV TROPISM AND ITS DETERMINANTS .................................................................................................14
PRO1ECT PURPOSE.................................................................................................................................17
METHODS ..................................................................................................................................................18
CLONING OF CHIMERIC VIRUSES INTO PSVIII VECTORS .....................................................................18
PCR AND SEQUENCE SCREENING...........................................................................................................19
DETERMINATION OF VIRAL TROPISM THROUGH INFECTIVITY ASSAYS...............................................20
RESULTS ....................................................................................................................................................26
DISCUSSION ..............................................................................................................................................38
BIBLIOGRAPHY.......................................................................................................................................41

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ACKNOWLEDGEMENTS



I would like to thank Dr. Paul Clapham Ior allowing me to work in his laboratory.
It has been a wonderIul experience and I have learned a lot Irom this introduction into the
research aspect oI Biology. The work I was able to perIorm in his lab has opened my
eyes to the possibilities oI what I may be able to do in the Iuture. In addition, I would
like to thank Dr. Paul Peters Ior his assistance in numerous laboratory assays, as well as
aiding me in problem solving on numerous occasions. Thanks also to Dr. Maria Duenas-
Decamp, Matthew Sullivan, Kathryn Richards and Catherine White Ior teaching me the
protocols I used in the lab, and providing me with the resources I needed to complete my
project. I would also like to thank Dr. Dave Adams Ior advising my project, helping me
initially contact Dr. Clapham, and providing timely and valuable inIormation throughout
my career here at WPI without which I would be very lost and uninspired.

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BACKGROUND


HIV Virus Overview
The Human ImmunodeIiciency Virus (HIV) is a retrovirus in the lentivirus
Iamily. A retrovirus is an enveloped virus with a single stranded positive RNA genome
(Clapham and McKnight, 2002). Common characteristics oI a lentivirus inIection
include: long incubation periods, persistent viral replication, destruction oI a particular
cell type, and neurological complications (Desrosiers and Letvin, 1987). HIV is most
commonly transmitted through blood, semen, vaginal Iluid and breast milk. The virus
itselI inIects cells oI the immune system, typically T-cells and macrophages, destroying
the ability oI the inIected individual to Iight oII the virus (Clapham and McKnight, 2001).
This destruction oI the immune system usually leads to AIDS (Acquired Immune
DeIiciency Syndrome) (Barre-Sinoussi, 1996).
A person with AIDS not only loses the ability to Iight oII the HIV virus, but also
becomes more susceptible to opportunistic inIections such as tuberculosis and pneumonia
(Barre-Sinoussi et al., 1983). According to the World Health Organization (WHO), over
2.5 million people were inIected with HIV in 2007 worldwide, and over 33.2 million
people are currently living with the HIV virus (World Health Organization, 2007).
Currently there are no cures or vaccines available Ior HIV, however a variety oI anti-viral
drugs are used to control the inIection, in combination therapies called HAART (Highly
Active Anti-Retroviral Therapy). HAART has reduced the number oI deaths Irom AIDS
in developed countries, but unIortunately this treatment does not work Ior everyone, and
it is not a cure.
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Genetics of HIV Infection
The HIV genome is single stranded RNA approximately 9 kb in size (HIV
Sequence Database, 2007). AIter HIV has entered the host cell, the RNA is transcribed
by the viral reverse transcriptase (RT) into a cDNA duplicate. This copy is integrated
into the host`s chromosomal DNA, and the viral proteins are then transcribed by the
cell`s own 'machinery. Although the HIV virus has a somewhat conserved sequence, it
can vary greatly because the RT enzyme lacks a prooI reading mechanism. As such, the
virus can mutate Irequently, oIten a mutation every 10,000 bases. The virus can mutate
so Irequently that a single inIected person can harbor many diIIerent strains oI the virus.
The HIV genome encodes nine diIIerent genes (Figure 1) that can be subdivided
into three categories: regulatory, structural and accessory.


Figure 1: The HIV-1 Genome (Harvard Images, 2006).

HIV accessory genes encode Iour proteins: Vpu, Vpr, ViI and NeI. The Iunctions oI the
accessory proteins range Irom the down regulation oI the CD4 receptor to aiding in the
release oI virus particles aIter replication (Turner and Summers, 1999). The regulatory
proteins are Tat and Rev. Rev induces expression oI the structural proteins, allowing the
virus to progress into the later phases oI the inIection. Tat aids in the transcription oI
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HIV mRNA Irom the integrated proviral DNA (Turner and Summers, 1999). The HIV
structural genes are: Gag, Pol and Env. The Gag gene is involved with recruiting viral
RNA to the cell membrane, as well as Iacilitating viral budding Irom an inIected cell
(Turner and Summers, 1999). The Pol gene produces a protease, a reverse transcriptase
and an integrase (Turner and Summers, 1999). Pol is required Ior newly budded virions
to mature into inIectious particles. The reverse transcriptase and integrase are involved in
the initial inIection oI the virus and integration oI its genome into the hosts. The Env
gene encodes two proteins that are incorporated into the envelope oI the virus. These
proteins Iorm the characteristic 'spikes oI the virus and aid the virus in identiIying and
inIecting its target cells (Turner and Summers, 1999).

HIV Envelope Structure and Function
Envelope Proteins
The HIV env protein is a glycoprotein created as a precursor protein (gp160) and
later cleaved into two Iunctional proteins, gp120 and gp41. The 'gp reIers to
'glycoprotein and the number reIers to the size oI the protein in kilodaltons (Turner and
Summers, 1999). The gp120 protein is expressed on the surIace oI the virus and is
primarily involved with the binding oI the receptors expressed on the target cell surIace.
Initially gp120 binds to CD4 which causes a conIormational change in the protein which
allowing the virus to bind to the secondary receptor or coreceptor.
The second envelope protein, gp41, is a transmembrane protein that contains a
hydrophobic domain that allows Iusion oI the viral membrane with the target cell
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membrane (Turner and Summers, 1999). The membrane Iusion allows core oI the virus
to enter into the cell`s cytoplasm initiating inIection oI the cells.

Structure of Jiral Envelope Proteins
The envelope spike is a complex oI proteins Iound on the surIace oI the virus that
is directly involved with receptor binding and membrane Iusion. The spike is made up oI
three gp120 proteins and three gp41 proteins, held together as a trimer (Clapham and
McKnight, 2002). Each gp120 protein has Iive variable loops in is structure (V1-V5)
(Figure-2); interspersed with more conserved domains (Clapham and McKnight, 2002).
Four variable loops (V1-V4) are exposed with disulIide bonds at their bases helping
maintain shape (Wyatt et al., 1998).


Figure 2: Structure oI the gp120 Core Element (Kwong et al.,
1998). Note: The gp120 used to produce this structure was
deleted Ior V1/V2 and V3, so the V1/V2 and V3 labels reIer to
just the bases oI these loops.

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Jariable Loops and Neutrali:ation Sensitivitv
The V3 loop has been implicated in viral tropism and co-receptor binding (Hwang
et al., 1991; Shioda et al., 1992; Cheesebro et al., 1996). One article by Shioda et al.
(1992) Iound that the V3 loop played a major role in coreceptor usage and viral tropism,
and limited amino acid substitutions in the V3 region could alter the coreceptor used and
the tropism oI the virus. Similarly, Hwang et al. (1991) also determined that the V3 loop
is essential Ior determining HIV-1 tropism. The V1/V2 loops vary in size and extent oI
glycosylation. These loops are targets Ior neutralizing antibodies. However variants with
altered V1/V2 sequences that are resistant to neutralizing antibodies rapidly emerge. The
V1/V2 loops sit over the coreceptor binding site and also protect the CD4 binding site
Irom neutralizing antibodies. gp120 is heavily glycosylated (50 carbohydrate by
weight), giving it Iurther protection against antibodies produced in response to the viral
inIection (Reitter at al., 1998; Losman et al., 2001).
The gp120 core structure consists oI two major domains, inner (leIt halI, Figure-
2) and outer (right halI). The inner domain consists oI two alpha-helices, a Iive stranded
beta-sandwich (brown in the Iigure) and several loops (Turner and Summers, 1999). The
outer domain is a stacked double barrel, with one barrel containing a six-stranded beta-
sheet which enIolds (middle portion oI the Iigure) and an alpha-helix as a seven-stranded
beta-barrel (upper right) (Turner and Summers, 1999).
The overall structure oI gp-120 is a heart shape, with the N and C termini on the
inner domain directed towards the virus, and the CD4 and chemokine receptor binding
sites directed towards the host cell (Kwong et al., 1998). CD4 binds in a carbohydrate-
Iree depression Iormed between the inner and outer domains. This cavity is Iilled with
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residues that do not interact with CD4. These residues have a high level oI sequence
variability, thus the CD4 binding surIace contains a large area oI non-CD4-binding
highly variable residues surrounded by the conserved CD4 binding region. This unusual
constantly mutating region oI non-CD4-binding residues may explain how the overall
CD4 binding site can evade potentially blocking immune responses initiated by HIV
inIection (Kwong et al., 1998).

b12 Antibodv Mav Protect Against HIJ Infection
A neutralizing human monoclonal antibody (b12) targets the gp120 CD4-binding
site oI HIV-1 (Parren et al., 1995). It has been shown experimentally to provide complete
protection against HIV inIection in SCID (Severe Combined ImmunodeIiciency) mice
used as an HIV model. Protection was achieved by passive immunization with
physiologically relevant doses oI b12 (Parren et al., 1995).
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Receptors and Fusion
In order Ior a cell to be inIected by HIV, the cell has to typically display two
speciIic receptors in order Ior the virus to bind and enter into the cell (Figure-3).


Figure 2: Attachment and Fusion oI HIV (Clapham and McKnight, 2002).


The major receptor used by HIV, CD4 (orange in the Iigure), is most commonly
Iound on the T-helper subset oI lymphocytes, macrophages and in some dendritic cells
(Knipe and Howley, 2001). The CD4 receptor Iunctions as an accessory receptor in a
cellular immune response to increase attraction between Helper T-cells and MHC class II
antigen presenting cells (Clapham and McKnight, 2002). Cell lines expressing high
levels oI CD4 are highly susceptible to HIV inIection. As such, CD4 expression is a
major determinant oI HIV tropism. When gp120 binds to CD4, a conIormational change
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occurs. The V3 loop protecting the binding site must move out oI the way. This means
the site is no longer being protected Irom the immune system by this variable region.
However, the V1/V2 loops have also been implicated in protecting the CD4 binding
region oI gp120 Irom antibodies aIter CD4 has been bound (Wyatt et al., 1998).
The two most common coreceptors used by the virus are CCR5 or CXCR4, which
are seven transmembrane (7TM) chemokine receptors (Clapham and McKnight, 2002).
Chemokines are hormones used in an immune response to inIections that aid in leukocyte
recruitment and other Iunctions. The mechanisms that determine what cell type a speciIic
viral strain inIects are not completely understood. However, research has shown that the
types oI receptors required Ior HIV to inIect the cell play a major role (Table 1)
(Clapham and McKnight, 2001). Typically HIV inIects cells displaying the CCR5
coreceptor, however variants that arise late in the disease utilize CXCR4 as a coreceptor.
HIV can also use other receptors in the same Iamily as CCR5 and CXCR4 as coreceptors.
However they are utilized less Irequently and there is no current evidence to indicate that
they inIluence replication in vivo or pathogenesis.
Over a dozen other coreceptors have also been identiIied that HIV can use in vitro
to inIect a cell, however only CCR5 and CXCR4 have been shown to be used in vivo
(Clapham and McKnight, 2001).





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Coreceptors Ligands Role Ior viral replication

In vitro In vivo
CCR1 MIP-1 , RANTES, MPIF-1, MCP-3
CCR2b MCP-1, MCP-2, MCP-3
CCR3 Eotaxin, Eotaxin-2, MCP-3, MCP-4, RANTES
CCR5 MIP-1 , MIP-1, RANTES, MCP-2
CCR8 I-309
CCR9 TECK
CXCR4 SDF-1
CX3CR1/V28 Fractalkine
STRL-33/BONZO/CXCR6 CXCL16
GPR1 ?
GPR15/BOB ?
APJ Apelin
ChemR23 Chemerin
RDC1 ?
Leukotriene B4 receptor Leukotriene B4
Table 1: Coreceptors used by HIV and SIV on CD4 expressing cell lines (Clapham and McKnight, 2001).

Individuals who are homozygous Ior a genetic mutation that Iunctionally
eliminates the CCR5 receptor are strongly protected against HIV-1 inIection (Liu et al.,
1996; Clapham and McKnight, 2001; Moore et al., 2004). This strongly illustrates the
importance oI the CCR5 receptor in vivo. However, individuals who have this mutation
are still susceptible and are occasionally inIected by CXCR4-using strains oI HIV (Moore
at al., 2004). A CXCR4 mutation has not been identiIied in humans because it is
believed the mutation would be lethal. Mice that lack the CXCR4 receptor do not survive
(Clapham and McKnight, 2001).
However, despite the coreceptor variance, once the CD4 receptor and the
coreceptor are bound to HIV, the HIV envelope proteins undergo another conIormational
change (panel C) that allows Iusion oI the viral membrane to the target cell membrane
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(panel D) (Clapham and McKnight, 2002). AIter membrane Iusion occurs, the virus
enters the cell (Turner and Summers, 1999).

HIV Tropism and its Determinants
Cell Tropism of HIJ
Some oI the Iirst cells to be inIected Iollowing transmission are mucosal cells in
the gut (Lim et al., 1993). Gut-associated lymphoid tissue (GALT) contains the majority

oI lymphoid tissue in the body (Guadalupe et al., 2003). CD4 memory T-cells in GALT
are depleted very rapidly during HIV inIection; however the surrounding tissues in the
periphery are slower to react to inIection. This tissue provides a signiIicant source oI
targets Ior the HIV virus. HIV also inIects the haematapoietic cells that express CD4 and
either CCR5 (R5) or CXCR4 (X4) coreceptors. In vitro research has shown that R5
viruses generally inIect both lymphocytes and macrophages. X4 strains predominately
inIect lymphocytes and T-cell lines which usually express CXCR4 but not CCR5 (Stent
et al., 1997). Initial inIections are almost always by CCR5 utilizing strains oI HIV,
however when X4 strains emerge during the course oI a patient`s inIection, their tropism
Ior T-cells is much broader than the R5 strains. This broader tropism opens up new cells
Ior the virus to inIect, allowing Ior the predominant virus to switch Irom R5 to X4 (Bleul
et al., 1997). SpeciIically, the X4 strains are very eIIicient at inIecting nave T-cells
which express CXCR4 but not CCR5 (Ostrowski et al., 1999). Dendritic cells have also
been implicated as a possible target Ior HIV inIection; however the extent to which
dendritic cells are inIected in vivo is still under debate. The current consensus is that
dendritic cells can support low levels oI HIV replication, and that they may play a role in
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transmitting virus Irom mucosal cells at the site oI transmission to T-cells in the lymph
nodes (Clapham and McKnight, 2001).
The emergence oI an X4 or R5X4 strain typically is associated with a more rapid
decline oI the immune system (Labrosse et al., 2001). However, some individuals can
progress to AIDS without the emergence oI an X4 strain. It is unclear whether R5
tropism broadens during the inIection by increasing HIV`s aIIinity Ior CD4 and/or CCR5
during the course oI inIection, or iI tropism is altered by other means. However, it has
been observed that 50 oI the people inIected with HIV have a switch in viral tropism to
X4 or R5X4 later in the disease.
Brain Tropism
The blood-brain barrier (BBB) protects the brain Irom the rest oI the body by a
system oI tight gap junctions between endothelial cells (Clapham and McKnight, 2001).
However, the HIV virus can bypass the BBB and inIect microglia and macrophages
within the brain relatively early in the inIection (Dallasta et al., 1999). This is most likely
accomplished by 'seeded cells, cells already inIected by the virus, entering the brain and
later releasing their viral load once past the BBB. Full length isolates or envelopes
derived Irom brain tissue conIer an enhanced tropism Ior CCR5 and CD4-expressing
macrophages and microglia cell lines containing low cellular levels oI CD4 and CCR5
(Clapham and McKnight, 2001). A possible explanation is that, since the brain is
relatively 'immuno-privileged, the lack oI antibodies in the brain may allow Ior viruses
that carry an envelope with a more open conIormation and better exposed CD4 binding
site to evolve. This may allow Ior a greater binding aIIinity between CD4 and the binding
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site within gp-120 on the virus, allowing the virus to utilize lower levels oI CD4 than it
would normally iI the binding site were in a more closed conIormation (Clapham and
McKnight, 2001).
HIJ Jiral Envelope Mav Determine Tropism
The exact mechanism that determines which coreceptor HIV utilizes and cell
types it can inIect is not Iully understood. However current research has shown that
sequence regions within the envelope genes can greatly inIluence viral tropism. Most oI
the research has pointed to regions within the gp120 protein, with important determinants
in the V1/V2 and/or V3 loops (Liu et al., 1990; O`Brien at al., 1990; McKnight et al.,
1995; Peters et al., 2004). The V3 loop in particular is a critical determinant oI coreceptor
use (McKnight et al., 1995; Peters et al., 2004). Other research has shown that regions
around the CD4 binding region can aIIect viral tropism (O`Brien et al., 1990).
Some slightly more controversial research has shown that regions within gp41 can
aIIect tropism (Glenn and Novembre, 2004). The gp41 protein does not have any direct
contact with any oI the receptors used Ior binding oI the virus to the target cell, however,
it may be possible that mutations within gp41 can cause conIormational changes within
gp120 that can aIIect tropism.
Our hypothesis was that neutralizing antibodies in vivo may select Ior envelope
sequences that protect critical nearby envelope sites (around the receptor regions) Irom
neutralization. However, these resistant envelopes may be compromised Ior their
capacity to interact with CD4. Hence, as neutralizing antibodies Iorm in vivo, this may
help induce a shiIt in tropism
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PRO1ECT PURPOSE

It has previously been shown that mutations in the gp120 and gp41 proteins can
aIIect the tropism oI HIV-1. In our laboratory, three viral strains (Q43-378.2, Q43-378.c,
and SQ43-380.1) oI HIV-1 removed Irom a single patient have shown varying viral
tropisms and sensitivities to various neutralizing antibodies (Peters et al., 2006). The
goal oI this MQP was Iirst to conIirm the tropism and neutralizing sensitivities oI the
three viruses by perIorming: viral titrations on TZM-bl and RC49 cell lines, and antibody
neutralizing assays. The project then aimed to create chimeric viruses, using the gp120
and gp41 proteins oI the three original viruses. The DNA sequences Ior these envelope
proteins were switched Ior those oI a diIIerent virus to determine the eIIects oI the gp120
and/or gp41 proteins on viral tropism and neutralization sensitivity. Also, we
investigated the possibility that an R5`s tropism may be modulated by neutralization
sensitivity. These chimeric viruses were transIected and tested in the same way as the
three original viruses. Understanding the determinants Ior HIV tropism and
neutralization sensitivity could lead to methods to block viral entry or to prevent switches
in tropism.

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METHODS
Cloning of Chimeric Viruses into pSVIII Vectors
Plasmid Preparations
Plasmid clones were produced Irom previously transIormed bacteria Irozen at
-80C as E.coli glycerol stocks. Bacteria containing plasmids were grown overnight
while shaking at 37C (pSVIII) or 30C (L4.1.1) in LB/AMP Broth Media. Plasmids
were isolated using the standard QIAGEN mini-prep protocol.

Plasmid Digestion
1 g oI plasmid was digested in a 50 L mixture (1 g oI plasmid, 1 L oI each
enzyme, 5 L oI NEBuIIer 4, and the remainder was Iiltered dH
2
0) Ior 1 hour at 37C.

Fragment Isolation
Digested plasmid was run on a 1 agarose gel, made with 1X TAE BuIIer and
0.5 g/ml Ethidium Bromide. The gel was run at 135 volts Ior 45 minutes. Desired gel
Iragments were cut out Irom the gel and run through the standard QIAGEN gel extraction
protocol. The Iinal mass oI puriIied DNA Iragment was quantitated by calculating its
optical density in a spectrophotometer.




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Fragment Ligation
300 ng oI insert DNA was mixed with 100 ng oI digested vector in addition to 1
L oI T4 ligase and equal portions oI 2x quick ligation buIIer. The mixture was
incubated at room temperature Ior 30 minutes to 1 hour.

Bacterial Transformation
4 uL oI ligated plasmid was transIormed into TOP10F` competent bacteria and
leIt on ice Ior 30 minutes. The bacteria were then heat shocked in a 42C water bath Ior
45 seconds, and then placed back on ice Ior an additional 2 minutes. 250 L oI S.O.C.
medium was added to the bacteria and the sample was then placed into a 37C shaker Ior
1 hour. The bacteria were then plated on LB/AMP plates and leIt to grow overnight at
37C.

PCR and Sequence Screening
PCR Screening
Initial screening oI cloned plasmids was done through PCR. Two primers were
used that Ilanked the MIeI site used to cut the plasmids during cloning. The PCR screen
checks Ior the inclusion oI both the gp120 and gp41 nucleic acid sequences in addition to
the proper orientation oI both sequences. The PCR mix included: 10 L oI 5x GoTAQ
BuIIer, 1 L oI 10 mM dNTP mix, 1 L oI 10 mM Primer 1, 1 L oI 10 mM Primer 2,
0.25 uL oI GotAQ Polymerase, and 36.75 L oI deionized dH
2
O per sample. The
thermocycler protocol was: 95C, 1:00 min; |95C, 1:00 min 35C, 1:00 min 72C,
2:00 min| X 35; 72C, 10:00 min; with a 4C hold. The two primers used were: V1/V2
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A3F (5` CCC ATA CAT TAT TTC C - 3`) and gp41 R1 (5` ATA GTG CTT CCT
GCT GCT C - 3`).

Sequence Screening
AIter conIirmation oI a possible chimera through PCR screening, the plasmid was
sequenced Irom the beginning oI the V1/V2 loop to the end oI gp160. The sequencing
mix was 4 L oI deionized dH
2
O, 2 L oI Big Dye, 2 L oI Primer, and 2 L oI plasmid
(~200ng/mL). The sequencing mix was run in a thermocycler using the Iollowing
protocol: 95C, 1:00 min; |95C, 1:00 min 55C, 1:00 min 60C, 6:00 min| X 28; with
a 4C hold. Three primers were used to sequence the plasmid: V1/V2 A3F (5` CCC
ATA CAT TAT TTC C - 3`), gp41 F1 (5` GAG CAG CAG GAA GCA CTA T - 3`),
and gp41 F2 (5` TGA ATA GAG TTA GGC AGG G - 3`). Following the protocol in
the thermocycler, 1 L oI 2 SDS was added to the mix. The entirety oI the mix (11 L)
was put in a hot bath at 95C Ior 5 minutes. AIter the 5 minute incubation, the samples
were run through DyEX puriIication columns, which required centriIugation at 2900
RPM Ior 3 minutes. The remaining solution was dried in a rotating dryer, and then sent
Ior sequencing by the CFAR sequencing Iacility.

Determination of Viral Tropism Through Infectivity Assays
Cell Lines and Maintenance
Three cell lines were used: 293T cells, TZM-bls, and RC49s. 293T cells, also
known as HEK 293T cells, are human embryonic kidney cells that allow Ior episomal
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replication oI transIected plasmids. 293T cells were split twice a week at a 1:17 ratio in
DMEM with 4 FBS.
TZM-bls, also known as JC53-bls, are HeLa cells containing high levels oI CD4,
CCR5 and CXCR4 receptors making it a excellent cell line Ior HIV inIection and
titration. TZM-bls contain a luciIerase reporter and a Beta-galactosidase gene controlled
by a promoter or LTR that allow Ior a color change Iollowing HIV inIection and tat
expression. TZM-bl cells were split twice a week at 1:8 in DMEM with 4 FBS.
RC49s are also HeLa cells, however they contain low amounts CD4 and high
amounts oI CCR5 mimicking that oI macrophages. Research has shown that RC49 cells
show similar sensitivity to HIV inIection as macrophages in vitro (Walter et al., 2005).
RC49 cells were split twice a week at 1:11 in DMEM with 4 FBS.

Passaging/Splitting
Cells were removed Irom plates using Versene Trypsin-EDTA. DMEM (4
FBS and 100 L oI concentrated Gentamycin) was used to neutralize the trypsin. The
DMEM 4 FBS and Gentamycin was also used as the standard media Ior the cells. HEK
293T cells were trypsinized Ior 30 seconds; HeLa cell lines (TZM-bl and RC49) were
trypinized Ior 4 minutes at 37C.

Cell Counting
Viable cells were counted using Trypan blue exclusion and a hemocytometer.
Cells were diluted with Trypan blue 1:10, then counted with the hemocytometer.

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Jiral Co-Transfection
Co-transIections were perIormed on 293T cells using calcium phosphate
according to Promega`s ProIection Kit. 293T cells were plated on 6-well plates at 2x10
5

cells/well. 48 hours aIter transIection, supernatant Irom transIected cells was harvested
and clariIied by centriIugation, and stored in liquid nitrogen until assay.

Fusion Assav
293T cells were cotransIected with pSVIII env and env
-
pNL4.3 using the
Promega calcium phosphate kit. TransIected 293T cells were washed twice with
Versene, then treated with Versene Trypsin-EDT to remove cells. Media was then
added to neutralize the trypsin and the cell suspension was placed into a 15mL conical
tube. The cells were centriIuged at 1200 RPM Ior 5 minutes. The supernatant was
removed and the cell pellet resuspended in 1mL oI media. Cells were counted and then
diluted to a concentration oI 1 x 10
5
cells/mL. 100 L oI the transIected 293T cells were
then added to a layer oI TZM-BL cells that were plated the day beIore at a concentration
oI 4 x 10
4
cells/well in a 48-well plate. The plates were then incubated Ior 5 hour at
37C. Cell Iusion was then scored based on the number and severity oI syncytia
Iormation.



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Jiral Concentration
Approximately 15 mL oI virus was concentrated using a 15mL Vivaspin Iiltration
tube at 2500 RPM Ior 35 minutes. Approximately 0.5 mL oI inIected media remained
aIter centriIugation. The media was resuspended with DMEM and stored at -152C.

Jiral Titrations
TZM-BL or RC49 cells (permissive Ior HIV-1) were plated at 2 x 10
4
cells/well
in a 48 well tray and leIt overnight. The next day the virus was thawed and serial ten-
Iold dilutions were made. Media was removed Irom the target cells and 100 uL oI each
virus dilution was added to the appropriate wells in duplicate. The cell and virus mix was
placed in an incubator at 37C and 5 CO
2
Ior 3 hours. 500 uL oI media was then added.
AIter 48 hours oI incubation, the cells were Iixed and stained (see below) depending on
target cell type, then counted by microscopy.

TZM-BL Fixing and Staining
Media was removed and cells were gently washed once with PBS.
Approximately 0.5 mL oI PBS/0.5 gluteraldehyde was added to each well Ior 10
minutes at 4C to Iix the cells. The Iixative solution was aspirated out and the wells then
rinsed once with PBS. 200 uL/well oI an X-gal/Yellow PBS mixture (1:80 dilution oI X-
gal:Yellow PBS, to make a Iinal concentration oI 0.5 mg/mL X-gal) was added. Yellow
PBS is PBS with 3 mM potassium Ierricyanide, 3 mM potassium Ierrocyanide, and 1 mM
magnesium chloride. The X-gal/Yellow PBS mixture was leIt on the wells overnight at
room temperature, and then removed the Iollowing day by aspiration. PBS/0.1 Azide
24
was added to keep the cells Irom drying out. InIected cells, stained blue, were counted
by light microscopy and the average number oI Iocus-Iorming units (FFUs)/mL were
determined. TZM-bls stain blue due to the presence oI the Beta-galactosidase gene.
When the host cell is inIected, the Beta-galactosidase gene is switched on by newly
synthesized tat. Following the inIection, when the host cell is in the presence oI X-gal, it
creates a blue product that is visible with light microscopy, marking an inIected cell.

RC49 Fixing and Staining
Media was removed and cells were washed in PBS and then were Iixed with 1:1
Methanol:Acetone (~0.5 mL/well Ior 8 minutes). Cells were rinsed with PBS and then in
PBS/0.1 Azide/1 FBS. 100 uL oI anti-p24 polyclonal antibody (1:40 dilution) was
then added to the cells and incubated at 37C Ior 1 hour. The wells were washed twice
with PBS/0.1 Azide/1 FBS. 100 uL oI the secondary antibody (1:400 dilution oI goat
anti-mouse B-galactosidase conjugate) was then added to each well, and the sample was
incubated Ior 1 hour at 37C. The wells were emptied and washed once with PBS/0.1
Azide/1 FBS, and then washed twice with PBS. 200 uL/well oI an X-gal/Yellow PBS
mixture (1:80 dilution oI X-gal:Yellow PBS, equaling 0.5mg/mL X-gal) was added. The
X-gal/Yellow PBS mixture was leIt on the wells overnight at room temperature and
removed the Iollowing day by aspiration. PBS/0.1 Azide was added to keep the cells
Irom drying out. InIected cells were stained blue and counted by light microscopy and
the average number oI Iocus-Iorming units (FFUs)/mL were determined. This alternate
Iixing and staining procedure had to be used because the RC49 cell line lacks the Beta-
galactosidase reporter gene that the TZM-bl cells contain. In this case, one antibody was
25
used to bind to an inIected cell; Iollowing this a second antibody conjugated to Beta-
galactosidase was added which to the Iirst antibody. Lastly, X-gal is added to the cells
which are turned blue by the Beta-galactosidase.

B12 Antibodv Neutrali:ation Assav
TZM-bl cells carrying the Beta-galactosidase and luciIerase reporter genes were
plated on 96-well plates at 8 x 10
3
cells/well and incubated overnight. Virus was thawed
and diluted to 8000 FFU/mL (200 FFU/well) on 48-well plates. The b12 antibody was
diluted to 200 ug/mL and then diluted Iurther using serial two-Iold dilutions on 48 well
plates. 30 uL oI each b12 dilution were added to separate wells oI a 96-well V-bottom
plate in duplicate. 30 uL oI virus was added to each well oI b12 and then the mix was
incubated at 37C Ior 1 hour. 50 uL oI each oI the antibody/virus mixtures was added to
the TZM-bl cells and then incubated at 37C overnight. The antibody/virus mixture was
aspirated, and 100 uL oI DMEM, 4 FBS was added to each well. The cells were
incubated Ior an additional 24 hours at 37C. AIter the incubation period the media was
aspirated and the luminescence mix was added (100 uL DMEM without phenol red and
100 uL oI Beta Glo Mix). The plates were leIt at room temperature Ior ~45 minutes to 1
hour, and were then read in a luminometer. For this assay, the luciIerase reporter gene
was used instead oI the Beta-galactosidase. The luciIerase mix simply lyses the cells
open and binds to a speciIic protein that was made by the inIected cells. This reaction
causes light to emit Irom the cells. Although this creation oI light is hard to detect by the
naked eye, it is easily detected and quantiIied by a luminometer.
26
RESULTS

Description of p43 Envelopes
HIV-1 envelopes used Ior this MQP were all derived Irom a single patient
designated p43. Patient-43 was categorized as an adult asymptomatic HIV

patient with
no signs oI neurological involvement. The envelope genes Irom this patient were derived
by nested PCR protocols Irom blood and semen (Peters et al., 2006). Three envelopes
Irom patient 43 were used in this study: 380.1, 378.2, and 378.c. The 378.c and 378.2
envelopes were ampliIied Irom the blood, while 380.1 was ampliIied Irom semen. All
envelope types were determined by Peters et al (2006) to be oI the R5 type (using CCR-5
and CD4 receptors in primary macrophage cells) and conIerred inIection oI cultured cell
lines containing high levels oI CD4. However, only envelope 380.1 was able to inIect
cells containing low levels oI CD4, and primary macrophages which are known to
express only low levels oI CD4 (Peters et al., 2006). In addition, 380.1 was previously
shown to be sensitive to neutralization by b12, a human monoclonal antibody which
binds the CD4 binding site on gp120. b12 has been the subject oI intense investigation as
the b12 epitope would have applications Ior a vaccine. Interestingly, 378.2 and 378.c
were previously shown to be resistant to b12.
Previous studies have shown that coreceptor usage can be mapped to the V3 loop
oI gp120 (Hwang et al., 1991; Shioda et al., 1992; Cheesebro et al., 1996). Variation in
amino acid residues oI the V3 loops can aIIect the overall conIormation oI the V3 loop,
playing a major role in determining ability oI HIV-1 to inIect T-cell lines and primary
macrophages (Shioda et al., 1992). The V3 loops oI the three envelopes used in this
27
MQP were aligned to check Ior patterns that could explain varying tropisms conIerred by
the envelopes (Figure 4).



Figure 4: V3 Loop Amino Acid Alignment of Parental Virus Envelopes 380.1,
378.2, and 378.c. Note: Boxes signiIy an amino acid residue that diIIers Irom the
380.1 sequence.


The V3 loop oI 378.c contains one amino acid diIIerence with 380.1, while 378.2
contains 2 amino acid diIIerences with 380.1.
Another theory was that truncations in the gp41 tail could cause changes in the
structure oI the gp41 and gp120 proteins, changing gp120`s binding kinetics to CD4 and
allowing it to utilize lower levels oI CD4 (Glenn and Novembre, 2004). This hypothesis
was also investigated by comparing amino acid alignments oI the gp41 cytoplasmic tails
compared to macrophage inIectivity (Figure 5). Previously published data indicated that a
truncation in the gp41 cytoplasmic tail may cause a conIormational change in both the
gp41 and gp120 proteins, allowing Ior a change in tropism.


Figure 5: Alignment of gp41 Cytoplasmic Tails of Parental Virus Envelopes.
28

It is unknown at this time which regions oI the three envelopes are involved in tropism
determination, so it was the goal oI this MQP to investigate these envelopes more closely
by creating chimeric viruses to determine the eIIects oI these gp120 V3 loops and gp41
sequences on viral tropism and neutralization sensitivity. Also, we investigated the
possibility that one oI the R5`s tropism may be modulated by neutralization sensitivity.

Construction of Chimeras
Patient-43 envelopes were previously cloned into pSVIIIenv plasmids. It was
determined that Iour chimeras would be made Irom the three original p43 envelopes
using an MIeI restriction site close to the junction between gp120 and gp41. However
due to the presence oI an additional MIeI site in some envelopes, it was not possible to
construct all possible chimeras. The chimeras that were created are shown in Figure 6.

Figure 6: Graphical Representation of Parental and Chimeric
Envelope Genes. Red denotes env 380.1, gray env 378.c, and green
env 378.2. The parental clones are shown in the upper halI oI the
Iigure, and represent three diIIerent cloned envelopes Irom HIV
patient-43. The vertical arrow denotes the position oI the MIeI site
used Ior subcloning.
29

Jerification of the pSJIII env Clones Carrving 380.1, 378.2 and 378.c Envelopes
Initially the three original envelope genes Irom HIV-1 patient-43 (380.1, 378.2
and 378.c) were cloned into plasmid pSVIII env via KpnI sites that Ilanked the env
sequences (Peters et al., 2006). VeriIication oI the pSVIII env clones was completed by
digesting with KpnI to check Ior appropriate band sizes. The digestion resulted in the
two expected bands, one oI ~2.9 kb and one oI ~5.1 kb (Figure 7).



Figure 7: Confirmation of Appropriate Plasmid and
Band Sizes of Three Original Cloned Envelopes.
Lower band at ~2.9 kb is gp160, upper band at ~5.1 kb
is the remainder oI the plasmid. The Iirst lane is a 1kb
ladder, whose lowest band is 0.5 kb not 1 kb; lane 3,
380.1; lane 4, 378.c; lane 5, 378.2.







Preparation of DNA Fragments for Chimera Construction
AIter conIirmation oI all three correctly cloned patient-43 env genes, the plasmids
were digested to remove gp41 or gp120 depending on which chimera was being created.
In order to isolate the gp41 Iragment, the plasmid was digested with MIeI, NcoI, and
NdeI; Ior the gp120 Iragment, the plasmid was digested with MIeI, NcoI and PacI. MIeI
cut between the gp120 and gp41 genes, whereas NcoI cut in the middle oI the pSVIII
vector, allowing Ior the two genes to be separated (Figure 8). However, the initial digest
produced bands oI approximately the same size, making separation by gel electrophoresis
very diIIicult. A third enzyme, either NdeI or PacI, depending on the digest, was added
30
to cut the vector in the undesired region oI the vector, producing two smaller bands and
one larger band; where the larger band contained the desired Iragment.




Figure 8: Restriction Map of Sites Used in the
pSVIII Plasmid Vector. gp41 extraction involved
using the MIeI, NdeI, and NcoI restriction sites,
producing bands oI approximately 4 kb, 3kb, 900 bp
and 300 bp, with the 4 kb band containing gp41.
gp120 extraction involved using MIeI, NcoI and PacI
restriction sites with bands oI approximately 5 kb, 2.6
kb and 600 bp. The 5 kb band contains gp120.


In all digests, the top band represented the desired gene. A typical digest is shown in
Figure 9.


Figure 9: Digestion of pSVIII Plasmid
to Isolate the gp41 or gp120
Fragment. Lane 1, 1kb ladder,
Lane 2, 380.1 gp120 digest (MIeI,
NcoI, PacI); Lane 3, 378.c gp120 digest
(MIeI, NcoI, PacI); Lane 4, 380.1 gp41
digest (MIeI, NcoI, NdeI); Lane 5,
378.c gp41 digest (MIeI, NcoI, NdeI).


Ligations to Form Chimeric Envelopes
Once the DNA Iragments were isolated Irom the gel, they were puriIied, ligated
in appropriate combinations, transIormed, and plated. Ampicillin-resistant bacterial
colonies Irom the plates were liIted and grown on a master plate. Miniprep DNAs
31
prepared Irom randomly selected colonies isolated Irom the master plate were screened
by PCR using primers that span the MIeI junction between gp120 and gp41 (Figure 10).

Figure 10: Confirmation of
Ligation by PCR Screen. PCR
screen oI the 378.c/380.1 chimera
using V1/V2 A3F and gp41 R1
primers. SuccessIul ampliIication
oI the chimera at ~1 kb (near the
second marker band Irom the
bottom). Lane 1, 1 kb ladder;
Lanes 2 and 19, dH
2
0; Lanes 3-
19, random samples; Lane 21,
positive control 380.1.

AIter conIirmation oI a correctly cloned chimera, the plasmid was sent Ior sequencing by
the CFAR sequencing Iacility. The DNA sequence was analyzed by aligning the two
original envelope genes to the chimera, and checking Ior the correct location oI the gp41
and gp120 sequences. AIter conIirmation oI the sequence, the chimeras were ready to be
transIected and tested.

Fusion Assavs on Chimeras and Original Envelope Genes
Co-cultivation oI env-expressing cells with cells expressing CD4 and an
appropriate coreceptor results in cell Iusion and syncytia production. These Iusion assays
were used to test whether chimeric envelopes were Iunctional Ior Iusion. Fusion assays
were perIormed by adding 293T cells transIected with each env viral clone on top oI a
layer oI TZM-bl cells containing CD4, CXCR4, and CCR5. Fusion was scored based on
the number and severity oI syncytia Iormation. A typical Iusion assay is shown in Figure
11.

32




Figure 11: Example Fusion Assay of Transfected 293T
Cells Placed on a Layer of TZM-bl Cells. Syncytia
staining Ior Iunctional envelope. 293T cells transIected with
plasmids encoding various envelop genes were mixed with
CD4 CCR5 TZM cells, and stained. Syncytia are visible
as large blue cell clumps. Picture generously provided by
Kathryn Richards.



All three original envelope genes and all the constructed chimeras scored as average
amounts oI Iusion (), whereas positive control NA20 B59 scored with high amounts oI
Iusion () (Table 2). These data show that the cloned chimeric envelop genes are all
Iunctional and exhibit similar inIection characteristics as the parental envelopes.
Virus Fusion
380.1 ++
378.2 ++
378.c ++
380.1/378.2 ++
380.1/378.c ++
378.c/380.1 ++
378.c/378.2 ++
NA20 B59
(positive control)
+++


Table 2: Fusion Assay Scoring for Original and Chimeric Envelopes.
The symbol signiIies no Iusion, signiIies low Iusion, signiIies
moderate amount oI Iusion, signiIies high amounts oI Iusion.

Titrations of Original and Chimeric Jiruses
Pseudo-typed viruses produced Irom 293T cells transIected with various env
genes were then titrated on TZM-bl cells that have high levels oI CD4, CCR5 and
CXCR4, and are permissive Ior inIection oI almost all HIV viruses. The viruses were
33
also all titrated on HeLa RC49 cells, which have low levels oI CD4 and high levels oI
CCR5. RC49 cells show similar sensitivity to HIV-1 R5 viruses as primary macrophages
(Walter et al., 2005). An example oI a typical titration is shown in Figure 12.

A B






Figure 12: Titrations of Viruses Infecting HeLa RC49 Cells After Staining. A) RC49 cells inIected and
stained at 10x magniIication. B) RC49 cells inIected and stained at 40x magniIication. The blue color indicates
virus inIected cells.

The TZM-bl cells containing all three receptor proteins were used as a positive control Ior
virus inIection (Iocus Iorming units per milliliter). InIection oI RC49 cells was then
expressed as a percentage oI the inIectivity observed the TZM-bl cells (Table 3).

Virus FFU TZM-bl FFU RC49
RC49 Infectivity
(% of TZM-bls)
380.1 3.95E+04 2.80E+03 7.09%
378.c NA NA NA
378.2 2.15E+05 1.70E+02 0.08%
380.1/378.c 4.85E+03 1.75E+02 3.61%
380.1/378.2 1.10E+05 1.45E+04 13.18%
378.c/380.1 1.38E+06 3.40E+04 2.46%
378.c/378.2 7.50E+05 2.70E+04 3.60%
NA20 B59 3.95E+05 3.55E+04 8.99%
Table 3. Titration Results for Eight Viruses Using TZM-bl and RC49 Cells.
The percentage oI RC49 cells inIected by pseudo-typed HIV was determined by
dividing the FFU RC49 by FFU TZM-bl and multiplying by 100. NA20 B59 is
the positive control.

34
We were unable to quantitate the Iocus Iorming units Ior the 378.c virus due to the
extremely low titer oI virus resulting Irom the transIection, despite numerous attempts to
concentrate and re-transIect the virus. Interestingly, the chimeras that contain either
gp120 or gp41 oI 378.c were Iunctional and titrated normally. All viruses carrying
gp120s Irom 380.1 and 378.c showed a strong inIection oI RC49 cells, at least 30 times
higher than non-macrophage tropic 378.2, and similar inIectivity to B59, a highly
macrophage tropic control envelope. In contrast, the capacity to inIect RC49 cells did not
correlate with a particular gp41 sequence.
These results thereIore suggest that both 380.1 and 378.c gp120s can conIer
cellular inIections via low levels oI CD4. In addition, env determinants Ior low CD4
inIection must be contained in gp120.

Antibodv b12 Neutrali:ation Assav
I next investigated the sensitivity oI viruses containing the various envelopes to
the b12 neutralizing antibody. The b12 monoclonal antibody is a human antibody which
binds an epitope overlapping the CD4 binding site on gp120. This creates a test to
determine whether the gp120 or gp41 proteins play a role in immune evasion, allowing
Ior better understanding oI what plays a role in antibody binding. Previously, envelopes
380.1 and 378.c were shown to be sensitive to the b12 neutralizing antibody, while the
378.2 virus was shown to be resistant (Peters et al., 2004).
A series oI b12 neutralization assays were perIormed to examine the sensitivity or
resistance oI each virus to the b12 antibody. The assay was perIormed on the three
original viruses and the chimeras (except the 378.c virus due to insuIIicient inIectivity).
35
Residual inIectivity was evaluated on HeLa TZM-bl cells using a luminescence reactant
(see Materials and Methods) whose signal is proportional to the amount oI HIV
replicating inside the cells. The Residual InIectivity was calculated as the current level
oI absorbance relative to the maximum luminescence reading when no b12 was present.
The residual InIectivity was plotted against b12 concentration (g/mL) (Figure 14).
36
A) Antibody b12 (anti-gp120) Neutralization of Parental Viruses.
0
20
40
60
80
100
120
0 0.20 0.39 0.78 1.56 3.13 6.25 12.5 25 50
B12 (ug/mL)
%

R
e
s
i
d
u
a
l

I
n
f
e
c
t
i
v
i
t
y
380.1 378.2 NA20 B59 VSV-G



B) Antibody b12 (anti-gp120) Neutralization of Chimeric Viruses.
0
20
40
60
80
100
120
0 0.20 0.39 0.78 1.56 3.13 6.25 12.5 25 50
B12 (ug/mL)
%

R
e
s
i
d
u
a
l

I
n
f
e
c
t
i
v
i
t
y
380.1/378c 378c/380.1 378c/378.2 380.1/378.2 NA20 B59 VSV-G


Figure 14: Antibody b12 Neutralization of Parental (A) and Chimeric (B) HIV Viruses. Summary oI
the b12 neutralization assay. All virsues start at 100 Residual inIectivity when no b12 is present and then
vary their viral inIectivity depending on their resistance or sensitivity as the amount oI antibody b12
increases. VSV-G, positive control; NA20B59, negative control.


37

Other than the positive control (VSV-G), the only other virus resistant to antibody b12 was
parental virus 378.2 (no chimeras containing 378.2 gp120 were tested). All other viruses
carrying gp120s Irom 380.1 and 378.c were neutralized by the b12 antibody with IC
50
s
between 0.78 and 50 g/mL. This data strongly suggests that the determinants Ior b12
sensitivity lie in gp120.

38
DISCUSSION

The major receptor used by HIV, CD4, is most commonly Iound on the T-helper
subset oI lymphocytes, macrophages, and in some dendritic cells (Knipe and Howley,
2001). Cell lines expressing high levels oI CD4 are highly susceptible to HIV inIection.
As such, CD4 expression is a major determinant oI HIV tropism. However, a coreceptor
must also be bound by HIV Ior inIection to be possible (Clapham and McKnight, 2002).
The mechanisms that determine what cell type a speciIic viral strain inIects are not
completely understood. However, research has shown that the types oI receptors required
Ior HIV to inIect the cell play a major role (Clapham and McKnight, 2001). Most oI the
research has pointed to regions within the HIV gp120 protein, with important
determinants in the V1/V2 and/or V3 loops (Liu et al., 1990; O`Brien at al., 1990;
McKnight et al., 1995; Peters et al., 2004). The V3 loop in particular is a critical
determinant oI coreceptor use (McKnight et al., 1995; Peters et al., 2004). Other research
has shown that regions around the CD4 binding region on gp120 can aIIect viral tropism
(O`Brien et al., 1990). From this data we hypothesized that neutralizing antibodies in
vivo may select Ior envelope sequences that protect critical envelope sites (around the
receptor regions) Irom neutralization. However, these antibody-resistant envelopes may
be compromised Ior their capacity to interact with CD4. Hence, the development oI
neutralization antibodies during inIection may help drive changes in tropism.
The three parental envelopes (378.2, 378.c, and 380.1) previously isolated by
Peters et al., 2006, exhibited varying macrophage tropisms even though they were all R5-
type envelopes. The 378.2 and 378.c envs tested to be non-macrophage tropic, while the
380.1 env showed to be macrophage tropic. Our results conIirmed these initial
39
classiIications done by Peters et al., 2006, with one exception. We were unable to create
a viable pseudotype Irom the 378.c envelope. The envelope appeared to be normal upon
sequencing, however it did not give rise to testable concentrations oI inIectious
pseudotype virions on transIection. However, the three parental gp120 and gp41 DNA
sequences were able to be cloned into three working chimeras. Notably, the 378.c/378.2
chimera, which contained envelope proteins Irom two viruses previously shown to be
non-macrophage tropic, in this project was shown to inIect cell type RC49 via low CD4,
and was thereIore macrophage tropic. Since the 378.2 chimera was shown in both studies
to be deIinitively non-macrophage tropic, it can be inIerred that the 378.c gp120 was
suIIicient to conIer macrophage tropism. This casts doubt upon the previous
classiIication by Peters et al., 2006, that 378.c was non-macrophage tropic.
The chimeric envelopes containing gp120s oI known macrophage tropic viruses
(380.1) exhibited high inIectivity in RC49 cells, suggesting that the gp120 is able to
conIer macrophage tropism. When the 380.1 gp120 was combined with a gp41 protein
Irom a non-macrophage tropic virus (378.2), the chimera still maintained high levels oI
RC49 inIection. This Iurther strengthened the theory that gp120 not gp41 conIers
macrophage tropism, while also suggesting that the gp120 protein is suIIicient to allow
inIection oI macrophages with no other changes. This data supports research that has
identiIied regions within gp120 that can conIer macrophage tropism (Liu et al., 1990;
O`Brien at al., 1990; McKnight et al., 1995; Peters et al., 2004).
The data Irom the b12 antibody neutralization, showed similar results to the viral
titrations oI the chimeras. The only virus previously shown to be resistant to b12 was
378.2 (Peters et al., 2006). This was conIirmed by our analysis oI the three parental
40
envelopes (Figure-14A). Due to limitations in the cloning process previously described
in the Methods, a pseudotype containing the gp120 oI the 378.2 virus was not created.
However, chimeras containing the gp41 oI 378.2 maintained their sensitivity to the
antibody, suggesting that the gp41 protein alone could not conIer resistance to the b12
antibody.
To Iurther investigate the theory that the gp120 protein conIers macrophage
tropism, more extensive chimeras should be created. Chimeras containing the gp120 oI
378.2 should be attempted again using alternative approaches (eg. overlapping PCRs).
Also, more chimeras should be created Irom the gp120 proteins oI macrophage tropic and
oI non-macrophage viruses to narrow down the region oI gp120 involved in tropism and
neutralization. Finally, point mutations could be introduced into the gp120s oI non-
macrophage tropic viruses (perhaps near the V3 loop or CD4 binding site) to identiIy iI
single amino acids are involved in tropism oI the virus, and to evaluate whether the
gp120 determinants that control tropism also direct neutralization sensitivity.
The project Iurther strengthens the hypothesis that the gp120 protein conIers the
macrophage tropism oI the virus, and the neutralization resistance to neutralizing
monoclonal antibodies. Although Iurther research has to be perIormed to identiIy the
exact regions oI the gp120 protein that cause changes in tropism, it can be saIely assumed
that the primary cause oI HIV-1 tropism and neutralization resistance lies within the
gp120 protein.
41
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http://www.hsph.harvard.edu/hai/images/laboratories/IigureHIV1and2.jpg

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