OF HIV-1 FITNESS ON DISEASE
PROGRESSION, SUBTYPE PREVALENCE, AND HOST-VIRUS EVOLUTION
Awet Abraha, Korey
Demers, Fred Kyeyune, Ryan Troyer, Immaculate Nankya, Kevin Ariën.
Collaborators: Sarah Ball, Miguel Quiñones-Mateu, Guido Vanham
1. Comparing ex vivo HIV-1 fitness to disease progression
Miguel has developed an HIV-1 dual infection/competition assay to measure
ex vivo fitness. We now have strong evidence that ex vivo HIV-1 fitness
is strong correlate and possibly a predictor of disease progression.
To expand these studies, Guido Vanham has sent us at least four consecutive
HIV-1 isolates spanning a period of two to five years of approximately
20 HIV-infected patients. We will measure the ex vivo fitness of each
isolate to compare with various clinical correlates of disease progression.
In a parallel study, Korey
is collecting samples from newly HIV-1 infected Ugandan and Zimbabwean
women participating in the HC-HIV study. Virus will be propagated from
these acute/early infections as well as during the course of disease
progression (2-5 years). Subtypes A, C, and D predominate in Uganda
whereas subtype C is exclusively found in Zimbabwe. Thus, we can use
these virus isolates to compare inter- and intrasubtype fitness as well
as the impact of subtype fitness on disease progression. Studies performed
by Sarah suggest that subtype C isolates may be significantly less fit
than subtype B isolates. This has lead to one of the most exciting hypothesis
in my career. Individuals infected with subtype C may have a slower
progression to AIDS than those infected with other subtypes. This possibility
coupled with enhanced subtype C transmission would explain the increased
prevalence of this subtype in the worldwide epidemic.
2. Examining a possible
relationship between HIV-1 fitness and quasispecies heterogeneity
The Red Queen Hypothesis states that a species must continue to evolve
into multiple quasispecies to retain or gain fitness advantage. Based
on this hypothesis, the continual increase HIV-1 quasispecies and genetic
diversity during the course of disease may be related to increased HIV-1
fitness. Kalonji will examine this relationship using the Belgium cohort.
3. Comparing the fitness
of different HIV types (-1 and –2), HIV-1 groups (M, N, and O),
and SIV isolates
This project will involve collaborations with the Vanham, Quinones,
and Arts laboratories. Kevin, a graduate student in Guido’s lab
will perform a pairwise competition experiment with the isolates of
the various primate lentiviruses, HIV types, and HIV-1 groups.
HIV-1 fitness to the env gene and using the env pseudotyping to measure
ex vivo fitness
Sarah clearly showed that efficiency of host cell entry was controlling
the dual virus competition. Thus, it appears that the HIV-1 env gene is
main determinant of fitness. Based on these findings, we are comparing
the fitness on env-pseudotyped HIV-1 isolates with propagated HIV-1 isolates
derived from the same patient samples.
Possible attenuation of HIV-1 during the epidemic (top)
HIV-1 RECOMBINATION AND DEVELOPMENT OF HETEROGENEOUS VACCINE
of fitness on the selection of intersubtype HIV-1 recombinants
In parallel to studies investigating subtype fitness, we also screened
and characterized intersubtype HIV-1 env recombinants. A low frequency
of recombination and possible hot sites for cross-overs suggest a limited
selection of intersubtype env recombinants similar to that observed in
vivo. This study will sub-clone the intersubtype env recombinants from
each dual infection into a HIV-1 molecular clone via the yeast cloning
method. These recombined clones will also be used in RANTES analog project
and in the subsequent design of a heterogeneous vaccine. HIV-1 clones
pseudotyped with these recombined env genes will be passaged in various
cell lines and primary cells to select for the most fit intersubtype recombinants.
We will then compare the fitness of these selected recombinants with the
fitness of the parental strains.
2. Generate and test
a construct containing intersubtype HIV-1 env recombinant as possible
This study will require extensive collaborations for small animal testing
and immunological assays. Adam will generate the constructs as described.
Mice will be vaccinated and boosted with the heterogeneous env DNA vaccine.
We will examine both humoral and CTL responses to various HIV-1 specific
epitopes of various subtypes.
examine the mechanisms of intersubtype HIV-1 recombination in vitro through
the use of reconstituted HIV-1 reverse transcription assays
Heather will clone several HIV-1 env DNA fragments into RNA expression
vectors. HIV-1 env RNA templates from different subtypes will then be
combined in reverse transcription assays. Sites of cross-overs during
reverse transcription and between RNA templates will be mapped and compared
to the recombination observed in dual infections. (top)
INVOVLED IN HIV-1 INHIBITION BY AND RESISTANCE TO CHEMOKINE ANALOGS
Erika Fraundorf, Dawn Moore, Fred Kyeyune.
1. Determine the mechanism
of HIV-1 inhibition by RANTES analogs
Based on results from Vince Torre, we suspect that RANTES analogs block
HIV-1 entry into a host cell by competitively binding to the CCR5 co-receptor.
Since different primary R5 HIV-1 isolates show variable susceptibility
to AOP-RANTES inhibition, it is unlikely that CCR5 receptor down-regulation
following AOP-RANTES binding could be the sole mechanism for HIV-1 inhibition.
Andre has cloned CCR5 with a mutation in cytoplasmic C-terminal that
prevents receptor internalization. U87 cells were stably transfected
with this mutant CCR5 construct. He is now accessing cell-surface receptor
expression, receptor-ligand interactions, and receptor internalization/recycling
by performing I125-RANTES binding assays and FACS analysis.
In future, we will compare RANTES analog inhibition of various primary
R5 HIV-1 isolates in the wt and mutant CCR5 cell lines. Erika has also
pseudotyped an HIV-1 laboratory clone with the env gene of several primary
HIV-1 isolates using the yeast cloning method. These viruses will propagated
from transfected cell lines and then used in drug susceptibility assays
2. Mapping the sites
conferring RANTES resistance in the envelope gene of R5 HIV-1 isolates
These studies will involve two approaches. In a previous study, Vince
identified a single amino acid residue (319) in the env V3 loop associated
with increased or decreased sensitivity to RANTES analog inhibition.
In addition, studies by Don Mosier’s group have identified an
adjacent amino acid residue (318) that was mutated in NNY-RANTES-resistant
HIV-1 isolates. Korey has used a site-directed PCR mutagenesis technique
to introduce these mutations in the env genes of two primary HIV-1 isolates.
An HIV-1 clone was then pseudotyped with these mutated env genes using
the yeast cloning method.
In future, these mutated viruses will then be propagated from transfected
cell lines and then used in drug susceptibility assays. We suspect that
these mutations will be associated with variable sensitivity to RANTES
The second approach to identify sites in the env gene associated with
resistance involves the intersubtype HIV-1 recombinants generated in
vitro. We have recombined an AOP-resistant and –sensitive R5 HIV-1
isolate by the dual infection technique. Recombined env genes were selected
by PCR as described. Similar to Adam’s project, the recombined
env genes are now being used to pseudotype the HIV-1 clone through the
yeast cloning method. Using multiple recombined HIV-1 strains in drug
susceptibility assays, we should be able to map the sites conferring
resistance to RANTES analogs.
1. Role of Vpr in AOP-RANTES stimulation of HIV-1 replication
Andre has previously demonstrated that high concentrations of AOP-RANTES
can stimulate replication of primary X4 HIV-1 isolates. This stimulation
appears to be mediated through a CCR5-mediated signaling cascade, ERK1,2
phosphorylation/activation, and increased HIV-1 integration. However,
it is unclear why AOP-RANTES stimulates only primary and not laboratory
X4 HIV-1 isolates. This discrepancy may be related to the dependence
on Vpr activity for the translocation of the HIV pre-integration complex
into the nucleus. In collaboration with Lou Mansky at Ohio State University,
we are now sequencing the Vpr gene to identify possible polymorphism
that may be related to this phenotype. In future, we may mutate the
Vpr gene in the HIV laboratory clones to determine if AOP-RANTES can
now stimulate virus production.
2. Introducing the natural
polymorphisms into the CCR5 ORF
3. Comparing sensitivity to
AOP-RANTES inhibition with HIV-1 fitness (top)
INVOLVED IN THE INITIATION OF HCV (-) STRAND RNA SYNTHESIS
Collaborators: Kathy Howard, Karl Venezia, Dr. Mary Barkley
This project is in its infancy
but is based on years of research into the mechanisms involved in the
initiation of HIV-1 reverse transcription. HCV like many other (+) stand
RNA viruses forms a complex RNA secondary structure at the 3’
end of RNA genome. This region may be synonymous in function with the
tRNA-viral RNA interaction involved in retroviral initiation of (-)
strand synthesis. Heather has cloned the HCV cDNA genome into an RNA
expression vector to transcribe various fragments of the 3’end
of the (+) strand HCV RNA. These RNA fragments are end-labelled and
probed with chemical or nucleases to determine RNA secondary structure.
In parallel, Karl and Kathy have cloned, expressed, and purified the
NS5b polymerase of HCV. They will be accessing the polymerase activity
of NS5b on the HCV RNA template. In addition, NS5b structure in the
presence or absence of HCV RNA will be probed using a protein footprinting
technique and mass spec. Heather will then footprint the RNA bound to
NS5b using the 3’ end of the (+) HCV RNA. Efficient initiation
of (-) strand RNA in this reconstituted assay may require the inclusion
of other viral proteins, e.g. NS4 or NS3 (protease/helicase). (top)