Phylogenetic Analysis of Population-Based and Deep Sequencing Data to Identify Coevolving Sites in the nef Gene of HIV-1

Poon, Art F. Y.; Swenson, Luke C.; Dong, Winnie; Deng, Wenjie; Pond, Sergei L. Kosakovsky; Brumme, Zabrina L.; Mullins, James I.; Richman, Douglas D.; Harrigan, P. Richard; Frost, Simon D. W.

doi:10.1093/molbev/msp289

Cited by 59 publications

(55 citation statements)

References 64 publications

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“…It is difficult to determine whether these inconsistencies are the result of technical errors or simply the failure to amplify, sequence, and detect consistently low-frequency variants. Variability between replicated UDS runs on the 454 platform has previously been reported (61,68). For example, Poon et al (61) found that many minority variants detected at levels between 1 and 5% in one replicate were undetectable in another.…”

Section: Discussionmentioning

confidence: 93%

“…Variability between replicated UDS runs on the 454 platform has previously been reported (61,68). For example, Poon et al (61) found that many minority variants detected at levels between 1 and 5% in one replicate were undetectable in another. Therefore, we believe that our filtering and background-calling procedure is unlikely to be the sole source of incongruent results between duplicate UDS runs and that other sources of noise could play a role, including PCR and sampling biases and various UDS instrument errors.…”

Section: Discussionmentioning

confidence: 93%

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Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Gianella

Delport

Pacold

et al. 2011

J Virol

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Reports of a high frequency of the transmission of minority viral populations with drug-resistant mutations (DRM) are inconsistent with evidence that HIV-1 infections usually arise from mono-or oligoclonal transmission. We performed ultradeep sequencing (UDS) of partial HIV-1 gag, pol, and env genes from 32 recently infected individuals. We then evaluated overall and per-site diversity levels, selective pressure, sequence reproducibility, and presence of DRM and accessory mutations (AM). To differentiate biologically meaningful mutations from those caused by methodological errors, we obtained multinomial confidence intervals (CI) for the proportion of DRM at each site and fitted a binomial mixture model to determine background error rates for each sample. We then examined the association between detected minority DRM and the virologic failure of first-line antiretroviral therapy (ART). Similar to other studies, we observed increased detection of DRM at low frequencies (average, 0.56%; 95% CI, 0.43 to 0.69; expected UDS error, 0.21 ؎ 0.08% mutations/site). For 8 duplicate runs, there was variability in the proportions of minority DRM. There was no indication of increased diversity or selection at DRM sites compared to other sites and no association between minority DRM and AM. There was no correlation between detected minority DRM and clinical failure of first-line ART. It is unlikely that minority viral variants harboring DRM are transmitted and maintained in the recipient host. The majority of low-frequency DRM detected using UDS are likely errors inherent to UDS methodology or a consequence of error-prone HIV-1 replication.Using standard population-based genotypic assays of HIV from individuals not yet treated with antiretroviral drugs, several studies (43,64,69,76) have estimated the rate of transmitted drug resistance to be between 8 and 27% in countries with the highest rates of antiretroviral therapy (ART) use. In resource-limited settings, in which the introduction of ART has been more recent, the estimated frequency of transmitted drug resistance mutations (DRM) is substantially lower (41). It appears, however, to be increasing in these settings as well (2,14,51). Using more-sensitive genotypic assays, different research groups (15,30,32,37,48,58,65) have reported higher proportions of transmitted DRM in ART-naive individuals. The clinical importance of these low-level DRM remains unclear, as they have been associated with clinical consequences in some (21,24,32,36,37,40,46,54,55,63,66,71) but not all (30, 47, 58) studies.Highly sensitive assays for detecting low-frequency DRM include point mutation assays and high-resolution sequencing techniques. Point mutation assays, such as allele-specific PCR, can detect DRM at frequencies as low as 0.01% of the sampled viral population (31,45,53,54), but they do not provide information about the sequence context surrounding a given DRM and may be prone to false positives at the lower level of detection (22). High-resolution sequencing techniques, such as single ge...

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 93%

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Gianella

Delport

Pacold

et al. 2011

J Virol

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“…Here, we made a number of simplifications. (i) Epistasis is known to exist for other RNA viruses (45)(46)(47)(48) and has been reported for HIV (49,50); it may contribute to the average values of LD. We hope that the importance of epistasis for the HIV genome in vivo will become clearer in the future, but we have ignored it here.…”

Section: Discussionmentioning

confidence: 96%

Estimate of effective recombination rate and average selection coefficient for HIV in chronic infection

Batorsky

Kearney

Palmer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

101

152

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HIV adaptation to a host in chronic infection is simulated by means of a Monte-Carlo algorithm that includes the evolutionary factors of mutation, positive selection with varying strength among sites, random genetic drift, linkage, and recombination. By comparing two sensitive measures of linkage disequilibrium (LD) and the number of diverse sites measured in simulation to patient data from one-time samples of pol gene obtained by single-genome sequencing from representative untreated patients, we estimate the effective recombination rate and the average selection coefficient to be on the order of 1% per genome per generation (10 −5 per base per generation) and 0.5%, respectively. The adaptation rate is twofold higher and fourfold lower than predicted in the absence of recombination and in the limit of very frequent recombination, respectively. The level of LD and the number of diverse sites observed in data also range between the values predicted in simulation for these two limiting cases. These results demonstrate the critical importance of finite population size, linkage, and recombination in HIV evolution.

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“…Sites V74, A83, and D175 lie within motifs implicated in the modulation of cell signaling pathways by Nef (20). While site S169 has no previously identified role in Nef function, a recent analysis showed that this site is coevolving with N157 and is therefore likely to have some functional role (51). The remaining six other sites under purifying selection (N52, A84, Y135, G140, H171, and V180) have no previously defined associations with known functions of Nef.…”

Section: Figmentioning

confidence: 99%

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

Lewis

Lee

et al. 2012

J Virol

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Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8؉ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo.

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Phylogenetic Analysis of Population-Based and Deep Sequencing Data to Identify Coevolving Sites in the nef Gene of HIV-1

Cited by 59 publications

References 64 publications

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Estimate of effective recombination rate and average selection coefficient for HIV in chronic infection

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

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