Towards an HIV-1 cure: measuring the latent reservoir

Bruner, Katherine M.; Hosmane, Nina N.; Siliciano, Robert F.

doi:10.1016/j.tim.2015.01.013

Cited by 170 publications

(211 citation statements)

References 111 publications

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“…S2), which is consistent with other reports [5,6,10,13]. A small proportion of sequences had stop codons not obviously due to G-to-A hypermutation (average 3%, range 0-12%) .…”

Section: Hypermutated Sequences Are Frequent In Hiv-1 Reservoirssupporting

confidence: 91%

“…The quantitative virus outgrowth assay (QVOA) represents the "gold standard" [4][5][6], but this assay underestimates the true size of the functional reservoir due to incomplete induction by PHA stimulation. Ho et al [7] showed that the functional HIV-1 reservoir may be 60-fold larger than originally estimated.…”

Section: Introductionmentioning

confidence: 99%

“…PCR based assays are also commonly used for quantifying the HIV-1 reservoir, but these assays overestimate the size of the functional reservoir because they cannot distinguish between replication-competent and defective viral genomes. Quantification of the HIV-1 reservoir by PCRbased methods typically give at least 100-fold higher numbers that the QVOA because of defective proviruses [4][5][6]. Many defective proviruses have large internal deletions [7,8].…”

Section: Introductionmentioning

confidence: 99%

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Establishment and stability of the latent HIV-1 DNA reservoir

Brodin

Zanini

Thebo

et al. 2016

Preprint

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HIV-1 infection currently cannot be cured because the virus persists as integrated proviral DNA in long-lived cells despite years of suppressive antiretroviral therapy (ART). To characterize establishment, turnover, and evolution of viral DNA reservoirs we deep-sequenced the p17gag region of the HIV-1 genome from samples obtained from 10 patients after 3-18 years of suppressive ART. For each of these patients, whole genome deep-sequencing data of HIV-1 RNA populations before onset of ART were available from 6-12 longitudinal plasma samples spanning 5-8 years of untreated infection. This enabled a detailed analysis of the dynamics and origin of proviral DNA during ART. A median of 14% (range 0-42%) of the p17gag DNA sequences were overtly defective due to G-to-A hypermutation. The remaining sequences were remarkably similar to previously observed RNA sequences and showed no evidence of evolution over many years of suppressive ART. Most sequences from the DNA reservoirs were very similar to viruses actively replicating in plasma (RNA sequences) shortly before start of ART. The results do not support persistent HIV-1 replication as a mechanism to maintain the HIV-1 reservoir during suppressive therapy. Rather, the data indicate that viral DNA variants are turning over as long as patients are untreated and that suppressive ART halts this turnover.

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“…S2), which is consistent with other reports [5,6,10,13]. A small proportion of sequences had stop codons not obviously due to G-to-A hypermutation (average 3%, range 0-12%) .…”

Section: Hypermutated Sequences Are Frequent In Hiv-1 Reservoirssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Establishment and stability of the latent HIV-1 DNA reservoir

Brodin

Zanini

Thebo

et al. 2016

Preprint

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“…These therapies are not curative, as latent, transcriptionally silent virus can spontaneously reactivate from sanctuaries and rapidly rekindle viral infection after withdrawal of therapy (1)(2)(3). Therefore, the molecular mechanisms of the activation and suppression of HIV transcription are of great interest.…”

mentioning

confidence: 99%

“…Tat is a small protein, typically found in a full-length form of ϳ101 amino acids (aa) 3 or as a splice variant (72 aa) encoded only by the first exon of the tat open reading frame. Both isoforms efficiently transactivate the HIV promoter in the 5Ј long terminal repeat (LTR).…”

mentioning

confidence: 99%

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7

Ali

Ramage

Boehm

et al. 2016

Journal of Biological Chemistry

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The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat.

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Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

et al. 2018

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IntroductionThe latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype‐tolerance using digital PCR.MethodsA subtype‐B‐specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype‐tolerance in digital PCR (Bio‐Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.Results HIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R 2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R 2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty‐nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.ConclusionsThe results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non‐B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

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Towards an HIV-1 cure: measuring the latent reservoir

Cited by 170 publications

References 111 publications

Establishment and stability of the latent HIV-1 DNA reservoir

Establishment and stability of the latent HIV-1 DNA reservoir

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

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