Possible involvement of distinct phylogenetic clusters of HIV-1 variants in the discrepancies between coreceptor tropism predictions based on viral RNA and proviral DNA.
BACKGROUND
The coreceptor tropism testing should be conducted prior to commencing a regimen containing a CCR5 antagonist for treatment of HIV-1 infection. For aviremic patients on long antiretroviral therapy, proviral DNA is often used instead of viral RNA in genotypic tropism testing. However, the tropism predictions from RNA and DNA are sometimes different. We examined the cause of the discrepancies between HIV-1 tropism predictions based on viral RNA and proviral DNA.


METHODS
The nucleotide sequence of theenvC2V3C3 region was determined using pair samples of plasma RNA and peripheral blood mononuclear cell DNA from 50 HIV-1 subtype B-infected individuals using population-based sequencing. The samples with discrepant tropism assessments between RNA and DNA were further analyzed using deep sequencing, followed by phylogenetic analysis. The tropism was assessed using the algorithm geno2pheno with a false-positive rate cutoff of 10 %.


RESULTS
In population-based sequencing, five of 50 subjects showed discrepant tropism predictions between their RNA and DNA samples: four exhibited R5 tropism in RNA and X4 tropism in DNA, while one exhibited the opposite pattern. In the deep sequencing and phylogenetic analysis, three subjects had single clusters comprising of RNA- and DNA-derived sequences that were a mixture of R5 and X4 sequences. The other two subjects had two and three distinct phylogenetic clusters of sequences, respectively, each of which was dominated by R5 or X4 sequences; sequences of the R5-dominated cluster were mostly found in RNA, while sequences of the X4-dominated cluster were mostly in DNA.


CONCLUSIONS
Some of HIV-1 tropism discrepancies between viral RNA and proviral DNA seem to be caused by phylogenetically distinct clusters which resides in plasma and cells in different frequencies. Our findings suggest that the tropism testing using PBMC DNA or deep sequencing may be required when the viral load is not suppressed or rebounds in the course of a CCR5 antagonist-containing regimen.
Department of Pharmacy, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan ; Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Hiroshi
H
Kotani
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Koji
K
Sudo
Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Naoki
N
Hasegawa
Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Hiroshi
H
Fujiwara
Department of Pharmacy, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan ; Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Tomohisa
T
Hayakawa
Department of Pharmacy, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan ; Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Osamu
O
Iketani
Department of Pharmacy, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Masaya
M
Yamaguchi
Department of Pharmacy, Keio University Hospital, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan ; Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, 105-8512 Japan.
Mayumi
M
Mochizuki
Center for Infectious Diseases and Infection Control, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Satoshi
S
Iwata
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan.
Shingo
S
Kato
CCR5-antagonisit; Coreceptor tropism; Deep sequencing; HIV-1; Proviral DNA; viral RNA