John West, PhD
RESEARCH INTERESTS:
Over the last nine years I have become interested in the mechanisms
of primate retroviral pathogenesis. Two broad areas within this
field have engaged my efforts. The first is the study of HIV evolution,
drug resistance and transmission, and the second is the structure
and function of the envelope glycoprotein and its relationship
with HIV-induced disease.
The mechanisms for and consequences of the development of drug
resistance in HIV-1 infection are of significant clinical and basic
science interest. The relationship between HIV-1 drug resistance
and replication fitness is unclear. Some studies have demonstrated
significant gains in virus fitness due the accumulation of drug
resistance whereas others have suggested that drug resistance is
accompanied by a loss in viral fitness. One of my primary interests
is to discern the relationship between antiretroviral resistance
and viral replicative fitness. In order to clarify the relationship
between resistance and viral fitness, I have utilized HIV-1 infection
of the SCID-hu mouse model. Two experimental designs have been
employed. In the comparative method viral strains, mutants and
isogenic variants for their capacity to replicate in SCID-hu grafts
and their ability to induce pathology (i.e. CD4 depletion). Utilizing
multiple biopsies, FACS and sensitive quantitative PCR techniques
we have been able to show that viruses containing accessory gene
deletions or those containing mutations in protease appear to exhibit
losses in fitness as a result of mutations. In contrast, many of
the reverse transcriptase inhibitor resistant strains do not appear
to cause loss of replicative capacity in our system.
The second experimental regimen focused on competition. SCID-hu
mice have been co-infected with isogenic viruses differing only
at a single point mutation in RT. The infected grafts are subjected
to multiple biopsies with analysis for pathology and viral load.
The DNA from each animal at each time point is then cloned and
sequenced or subjected to Taqman(r) detection to determine the
frequency of each viral allele in the tissue. Fitness calculations
have been used to determine whether the drug-resistant mutants
are more or less fit than wild type or each other. Interestingly,
we have found it extremely difficult to co-infect grafts. In our
experiments there appears to be a 'founder effect' where only one
of the simultaneously injected strains establishes itself. It is
not yet clear whether this is a technical problem or whether it
may correlate with the seeming reversion to homogeneity of the
HIV quasispecies in patients who have acquired the virus by IV
injection. Evidence from co-infection studies designed to study
viral recombination has also suggested a 'founder effect' in SCID-hu
mice as well as PBMC.
The second major focus of my work has been to assess the replication
and pathogenicity of longitudinal viral isolates from patients
on multi-drug regimens using the SCID-hu mouse model. We are focusing
on patients whose clinical data appears to imply phenotypical alterations
due to changes in envelope glycoprotein function rather than in
RT or PR. In addition we are also evaluating a number of viruses
from a Zambian cohort of discordant couples with particular emphasis
on the effects of promiscuous chemokine receptor usage and viral
replication and pathogenesis. Our current data suggests that we
can faithfully model the R5 to X4 co-receptor switch in our system.
The SCID-hu model, therefore, has allowed us the ability to investigate
and manipulate human disease in human tissue.
The studies focusing
on Env integrate well with my graduate training where I investigated
the role of the cytoplasmic domain in HIV-1 assembly. Specifically
we examined whether endocytosis of the HIV-1 envelope played
a role in regulating the incorporation of Env into virus particles.
We determined that alteration of the HIV-1 Env endocytosis signal
was insufficient to overcome defects in incorporation due to
mutation of the viral matrix (MA), thus, supporting the hypothesis
that incorporation requires a specific interaction between MA and
Env. Finally, regarding Env protein structure, we have reassessed
the structure of the SIV membrane-spanning domain (MSD). Through
the use of sequential truncation mutations we were able to define
the minimal C-terminal requirements in gp41 for SIV infectivity,
for cell-cell fusion and for glycoprotein transport and incorporation.
We show that the minima for each resides within distinct sequences
previously thought to lie within the MSD. We have proposed an
alternative model for the organization of the SIV MSD based on
our findings.
It is my intention to continue to investigate retroviral assembly
and pathogenesis with particular emphasis on the envelope glycoprotein
and its receptor. Understanding the qualities of glycoprotein that
potentiate virus transmission and selection early in infection
is a major focus. I am also interested in the potential role of
the cytoplasmic tail of Env in altering signal transduction pathways
in infected cells. I am continuing to try to understand what specifies
the interaction between viral Gag and Env and what specifies the
site of viral budding and release. Finally, I am interested in
the mechanisms and consequences of viral evolution and recombination
in the face of drug selection.
Publications:
West J and Wood C. 2003. The Role of Kaposi's Sarcoma-Associated
Herpesviruses/Human Herpesvirus-8 Regulator of Transcription Activation
(RTA) in Control of Gene Expression. Oncogene, 22:5150-5163
Brayfield, B., S. Phiri, C. Kankasa, J. Muyanga, H. Mantina, G.
Kwenda, J. T. West, G. Bhat, D. Marx, W. Klaskala, C. Mitchell,
and C. Wood. (2002) Postnatal human herpesvirus-8 and human immunodeficiency
virus-1 infection in mothers and infants from Zambia. J. Inf. Dis.
187:559-68.
West J. T., S. K. Weldon, X. Lin, S. Wyss, M. Thali, and E. Hunter.
(2001) Mutation of the dominant endocytosis motif in HIV-1 gp41
can complement matrix mutations without increasing Env incorporation.
J. Virol. Submitted for publication.
West J. T., P. B. Johnston, S. R. Dubay and E. Hunter. (2001)
Mutations within the putative membrane-spanning domain of the SIV
transmembrane glycoprotein define the minimal requirements for
fusion, incorporation and infectivity. J. Virol. (under review).
Salzwedel, K., J. T. West and E. Hunter. (1999) A conserved tryptophan-rich
motif in the membrane-proximal region of the human immunodeficiency
virus type 1 gp41 ectodomain is important for Env-meidated fusion
and virus infectivity. J. Virol. 73(3): 2469-2480.
Salzwedel, K., J. T. West, M. J. Mulligan and E. Hunter. (1998)
Retention of the HIV-1 envelope glycoprotein in the endoplasmic
reticulum does not redirect virus assembly from the plasma membrane.
J. Virol. 72(9): 7523-7531.
Fultz, P. N., L. Su, P. May and J. T. West. (1997) Isolation of
a Sooty Mangabey simian T-cell leukemia virus type I [STLV-I(sm)]
and characterization of a mangabey T-cell line coinfected with
STLV-1(sm) and simian immunodeficiency virus SIVsmmPBj14. Virology.
235: 271-285.
|
