CFAR Developmental Pilot Awards – Current Recipients

Dan Bolon, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Pharmacology
Funding Period:  November, 2011-present

Title of Research Project
Systematic Mutagenesis and Fitness Analyses of HIV

ABSTRACT 
Selective pressures dramatically influence the HIV genome. For example, drug
therapy places strong selection pressure on the RT and PR genes, while
substitutions selected to combat host immunity also influence fitness. All genes
in HIV are further constrained by selection pressure to maintain function.
For example, mutations in the envelope that destroy the ability to dock with
host receptors will be strongly selected against. Despite the important role of
selective pressure in HIV, it has been challenging to investigate the fitness
effects of individual mutations in HIV. We have developed an approach to
systematically generate all possible point mutants in an otherwise identical
genetic background; and to interrogate the fitness effect of each point
mutant. This approach (termed extremely methodical and parallel investigation
of randomized individual codons; EMPIRIC) uses deep sequencing to monitor
the abundance of each mutant in a mixed population and has been extensively
vetted using yeast as a model system. Drs. Bolon and Clapham are focused on
the CD4 binding loop of gp120, a critical early contact site for CD4. Individual
substitutions in the variable N‐terminal region of the loop, as well as in
conserved CD4 contact residues, will be investigated following selection for
T‐cell and macrophage infection, as well as after neutralization by the potent
CD4bs mab, VRC01. This project will provide extensive information on the
functional contribution of individual residues in the CD4 binding loop, an
envelope site that is also a major target for vaccines. This project will also
introduce and assess a systematic mutagenesis approach that can be applied
to any of the HIV proteins to study their functions and their interactions with
host proteins. We anticipate that this project will serve as the basis of a
long‐term program to understand the relationship between HIV sequence
and fitness as it pertains to human health.


Maria Jose Duenas Decamp, Ph.D.
Instructor
Program in Molecular Medicine
Funding Period:  November, 2011-present

Title of Research Project
HIV-1 Hematopoietic Stem/Progenitor cell Differentiation and Tropism
Implication for HSPC Infection

ABSTRACT
Human Immunodeficiency Virus (HIV-1) infection is characterized by gradual
immune system collapse and haematological abnormalities, such as neutropenia,
thrombocytopenia and anemia. HIV-1 enters cells through the binding of gp120
to CD4 receptor and CCR5 and/or CXCR4 coreceptor.  Macrophage-tropic virus
(R5 mactropic) and non-macrophage-tropic R5 viruses (non-M-tropic) can infect
via CCR5 coreceptor, while T-cell tropic viruses (T-tropic) enter via CXCR4, and
dual-tropic viruses via CCR5 and/or CXCR4. Hematopoietic stem cells (HSC) can
renew themselves or differentiate to a variety of specialized cells such as
lymphocytes, macrophages, erythrocytes, etc. Subsets of hematopoietic stem
and progenitor cells (HSPCs) express HIV receptors CD4 and CCR5 or CXCR41.
We will test R5 viruses from different tissues, and T-tropic and dualtropic
primary isolates derived from maraviroc treated-patients obtained at different
time points after treatment,to determine their capacity to infect HSPC.
Until now, the extent that HIV-1infection in HSCs and hematopoietic progenitor
cells (HPC) results in the establishment of long term viral reservoirs in infected
individuals remains unknown. Although there is direct evidence that HIV can
target and integrate in hematopoietic stem and progenitor cells (HSPC), it is
unknown if HIV+ HSPCs can develop into live HIV-infected CD8+ and CD4+ T
cells that are capable of releasing and transmitting viral infectious particles.
The aims of this grant are:
(1) to evaluate the infectious activity of mac-tropic and non-mac-tropic R5
viruses and primary isolates obtained from maraviroc treated-patients; and
(2) to determine if HIV-infected HSPCs can differentiate to HIV+ T cells.

Katherine Fitzgerald, Ph.D.
Associate Professor
Department of Medicine, Division of Infectious Diseases & Immunology
Funding Period:  November, 2011-present

Title of Research Project
Innate Immune Recognition of HIV DNA

ABSTRACT
Mutations in the human gene encoding TREX1 cause autoimmune diseases that
resemble systemic lupus erythematosus. TREX1 deficiency in mice leads to the
accumulation of cytosolic DNA from endogenous retroelements, which trigger
type I interferon production and autoimmune pathology. Interestingly, TREX1
also degrades HIV DNA generated during HIV-1 infection and thereby prevents
triggering of innate immunity. It has been shown recently that if TREX1 is
absent then HIV DNA accumulates in the cytosol and triggers a type I IFN
response, allowing the virus to be detected. Manipulation of these events
may allow early detection of HIV thus preventing dissemination in infected
individuals. Recognition of DNA has emerged as a key trigger of innate
defense against other viruses. The DNA-sensing pathway involved in sensing
DNA derived from endogenous retro-elements or from HIV is known to involve
the ER resident protein STING, which couples DNA recognition to activation of
the kinase TBK1, that in turn phosphorylates and activates the transcription
factors IRF3/7 to turn on type I IFN gene transcription. However, the receptor
that binds DNA in these scenarios is unknown. The Fitzgerald lab has studied
the molecular basis for the immune stimulatory activity of DNA, work which
has led to the identification of several novel DNA receptors and signaling
molecules important in innate anti-viral immunity. We have also found that
DNA sensing pathway recognizes host DNA from endogenous retro-elements.
Using mass-spectrometric analysis, two novel DNA binding proteins, IFI16
and DDX41, were identified, which we have shown associate with STING
and thus represent exciting candidates which could could mediate cell intrinsic
responses to DNA during HIV infection. The hypothesis to be explored in this
study is that IFI16 or DDX41 act as cytosolic sensors for HIV DNA and turn on
key protective anti-viral type I IFN responses in situations where TREX1 is
defective. We propose that these newly identified candidate sensors are
responsible for the cell-intrinsic type I IFN response to cytosolic retro-viral
DNA. Moreover, we will also try to define a role for SAMHD1, a recently-
described regulator of the DNA-sensing pathway.

Paul Kaufman, Ph.D.
Professor
Program in Gene Function and Expression
Funding Period:  November 2011-present

Title of Research Project
Optimization of high-throughput screening for novel candidate anti-fungal compounds using AIDS-patient-isolated Candida albicans strains

ABSTRACT
Adhesion to surfaces and biofilm formation are crucial steps during pathogenesis by
fungi such as Candida albicans, which is especially dangerous to immuno-
compromised AIDS patients. We have identified a family of related small molecules
that inhibit adhesion of C. albicans to polystyrene surfaces. We plan to use these
compounds to develop improved assays suitable for high throughput screening,
and also to optimize secondary assays measuring interactions with human cells.
Together, these efforts will leverage our existing data to the point where we can pursue
outside funding.

Andrei Korostelev, Ph.D.
Assistant Professor
Biochemistry and Molecular Pharmacology
RNA Therapeutics Institute
Funding Period:  November, 2011-present

Title of Research Project
Structural Basis for Translational Regulation of HIV-1 Replication

ABSTRACT
Replication of HIV-1 depends on the precursor polyprotein Gag, which mediates the
assembly and release of the virus. Translation of the gag gene in host cells relies on
the internal ribosome entry site (IRES), which is a structured RNA sequence in the
5´untranslated region of the HIV-1 genomic RNA. Several viral IRESs have been
studied to date, with recent molecular structures significantly advancing our mech-
anistic understanding of how viruses hijack the host’s translational apparatus. By
contrast, no structural information exists for the HIV-1 IRES, which likely employs a
mechanism distinct from those of other viral IRESs. I propose to use X-ray crystal-
lography and cryo-EM to elucidate the structural basis for IRES-dependent
translation of Gag. This study will bring insights into the translational regulation
of HIV replication, and may pave the way for development of novel anti-HIV
therapeutics.

Evelyn Kurt-Jones, M.D.
Professor
Department of Medicine, Division of Infectious Diseases & Immunology
Funding Period:  November, 2011-present

Title of Research Project
CD200:CD200R1 Interactions in Kaposi Sarcoma Infections

ABSTRACT
Kaposi’s Sarcoma (KS) is a HIV-associated malignancy that is caused by the human
herpesvirus 8 (HHV8) double-stranded DNA virus. HHV8 disease is still prevalent in
HIV-infected individuals worldwide despite the successes of antiretroviral therapy,
and can be manifest as KS, multicentric Castleman’s disease, or primary effusion
lymphoma. KS is characterized by angioproliferative multifocal tumors of the skin,
mucosa, and viscera. KS lesions are comprised of both distinctive spindle cells of
lymphatic endothelial origin and an inflammatory infiltrate composed of macro-
phages, dendritic cells, lymphocytes and also plasma cells. KS is considered a
prototype for the interplay between inflammation, cancer, and viral infection,
in which intense inflammation may drive the progression of oligo or polyclonal
lesions to evolve into multifocal lesions, each of a monoclonal origin. The host
immune response includes secreted factors that recruit effector cells (inflam-
mation) and factors that limit virus growth (antivirals including interferon).
How HHV8 and HHV8-infected cells avoid the anti-viral mechanisms directed
against it during the intense host inflammatory response are poorly understood,
but play a key role in the evolution of disease. The viral K14 open reading
frame protein is a lytic phase ortholog of the mammalian CD200 protein. CD200
is a transmembrane protein that is expressed onthe surface of endothelial cells,
neurons, and other cells. It interacts with its receptor CD200R1 that is expressed
on the surface of macrophages, dendritic cells, and other myeloid cells to down-
regulate the proinflammatory signaling and biological responses in these cells.
The HHV8 K14 open reading frame gene (ORF) product is a lytic phase protein
that is a structural (40% amino acid identity) and functional ortholog of the
mammalian CD200 protein. The hypothesis of this proposal is that the HHV8
K14 ORF protein (viral, or vCD200) interacts with host CD200R1 receptors
and selectively down-regulates key anti-viral programs inmacrophages. We
propose that the ability of K14 vCD200 to regulate host innate immunity
results in an increase in virus replication. Thus, CD200R1 engagement by
vCD200 creates a permissive environment for virus replication in CD200R1
expressing myeloid cells.

Mary Munson, Ph.D.
Associate Professor
Biochemistry and Molecular Pharmacology
Funding Period: November, 2011-present

Title of Research Project
The Role of Host Exocytic Trafficking Machinery in HIV Assembly and Budding

ABSTRACT
Formation of infectious HIV-1 particles requires proper targeting and assembly
of the Gag polyprotein p55Gag (consisting of the four major structural proteins:
matrix, capsid, nucleocapsid, and p6), as well as trafficking of the Envelope
(Env) glycoprotein, to specific sites on the plasma membrane. The molecular
mechanisms behind Gag and Env targeting and assembly are poorly understood,
and include Gag myristoylization for membrane anchoring, Gag oligomerization,
and Env trafficking and processing in the host secretory pathway to the plasma
membrane. We propose that host exocytic processes are intimately involved
with promoting Gag and Env targeting and assembly at the plasma membrane,
either directly, or indirectly through trafficking of interacting factors. The core
of the exocytic (and recycling) trafficking machinery at the plasma membrane are
the exocytic SNARE proteins, and their regulator, the exocyst complex. We
hypothesize that disruption of function of the exocytic SNARE proteins and the
exocyst complex will lead to loss of assembled Gag at the plasma membrane
and therefore prevent viral assembly and budding. As such, these factors would
be potential new targets for anti-HIV therapy, and because these are host cellular
proteins, the virus would be unable to develop drug resistance against this new  
therapy.