Paper # 478
Molecular Cloning and Biological Characterization of Full-length Transmitted/Founder Viruses from Injection Drug Users, MSM, and Heterosexuals
Hui Li*1, K Bar1, C Tremblay2, J-P Routy3, B Keele4, F Gao5, M Markowitz6, B Haynes5, B Hahn1, and G Shaw1
1Univ of Alabama at Birmingham, US; 2Univ of Montreal, Canada; 3McGill Univ Hlth Ctr, Montreal, Canada; 4SAIC-Frederick, NCI-Frederick, MD, US; 5Duke Univ Med Ctr, Durham, NC, US; and 6Aaron Diamond AIDS Res Ctr, The Rockefeller Univ, New York, NY, US
Background: Single genome amplification (SGA)
followed by direct amplicon sequencing of plasma vRNA allows for the
unambiguous identification of transmitted/founder (T/F) viruses responsible for
productive HIV-1 infection. Here, we used SGA to identify and molecularly clone
T/F viruses resulting from 3 different modes of transmission: intravenous drug
use (IDU), men having sex with men (MSM) and heterosexual sex (HSX).
Methods: Full-length sequences of T/F viruses were
inferred from plasma vRNA by SGA analysis of overlapping 5’ and 3’ half genomes
and Long Terminal Repeats. The full-length proviral T/F genome was chemically
synthesized in 3 concatenating pieces or amplified from the peripheral blood
mononuclear cell vDNA in 2 overlapping pieces, with a unique restriction site
in the overlapping region. The fragments were cloned into the pCRXL-TOPO
plasmid vector and sequence confirmed to match the exact nucleotide sequences
of the T/F viruses.
Results: A total of 15 T/F proviral clones were
generated and all contained the expected 9 canonical gene open reading frames
intact. Following transfection into 293T cells, progeny viruses were infectious
in JC53BL cells and in human peripheral blood mononuclear cells. The majority
of the T/F viruses showed CCR5 co-receptor usage although 3 utilized X4, Bonzo,
or an as yet undetermined co-receptor. T/F viruses replicated efficiently in
CD4+ lymphocytes, but generally not in monocyte-derived
macrophages.
Conclusions: Infectious molecular clones of T/F
viruses that are responsible for productive clinical infection were generated
from HIV-1 subtypes A, B, C, and D, and such viruses captured the unique
infectivity properties associated with the earliest transmission events. A
spectrum of cell tropism and co-receptor specificities were observed in these
viruses, indicating that such diversity is present at the moment of virus
transmission and is not an acquired or adaptive. Analysis of T/F viruses can
make an important contribution to HIV-1 transmission, natural history, and
vaccine research.
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