Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART.

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load

Viral Decay Kinetics in the Highly Active Antiretroviral Therapy-Treated Rhesus Macaque Model of AIDS

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2–58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans

Phylogenetic diversity of low-level viremia.

<p>For each RM, phylograms represent RT-SHIV LLV sequences that were obtained between weeks 18 and 50 PI. Sequences were derived by SGA using plasma vRNA samples that were collected after maximal viral load suppression (<50 vRNA copies/mL) was initially achieved. There were a total of 437 nucleotide positions in the alignment encoding reverse transcriptase amino acids 65 to 210. Neighbor-joining phylograms were constructed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone-0088258-g002" target="_blank">Figure 2</a> and were rooted on the consensus sequence of the RT-SHIV inoculum (open black circle). Each asterisk denotes that the indicated LLV sequence contained a single mutation which was not observed in the pre-suppression variant profile. The identity of these mutations has been annotated on the phylograms in black lettering. Drug resistance mutations to compounds which were not used in this study are annotated in red lettering to the right of LLV sequence in which they were observed. These mutations are associated with resistance to the NNRTI Nevirapine (S162N and Q174R) and the NNRTIs Etravirine and Rilpivirine (E138K). Finally, sequences pertaining to the putative predominant plasma clone sequence identified in RM Mmu 37969 have been annotated “PPC”. These sequences were observed at four separate time points (weeks 34, 45, 47, and 50 PI).</p

RT-SHIV mutations with putative selective pressures^a.

a<p>Capital letters indicate the following RMs: (A: Mmu 38202; B: Mmu 37969; C: Mmu 37774; D: Mmu 38560; E: Mmu 38606). In the category “#≥5%” bold RM designations separated in parentheses specify that the indicated mutation was observed in <5% of sequence reads.</p>b<p>Observed by our group in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-VanRompay1" target="_blank">[46]</a>.</p>c<p>Observed in RT-SHIV_mne infected pigtail macaques (Shao et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-Shao1" target="_blank">[47]</a>).</p

Longitudinal analysis of plasma viral loads.

<p>Plasma viral loads were determined by TaqMan RT-qPCR using our standard viral load assay. All RMs began HAART (4 NRTI, 1 NNRTI) after eight weeks of infection. Rhesus macaques Mmu 37969 and Mmu 38202 were necropsied during therapy at weeks 50 and 52 PI respectively. Viremia rebounded in RMs Mmu 38560, Mmu 38606, and Mmu 37774 upon cessation of therapy at week 50 PI. These RMs were necropsied on the following weeks PI: (Mmu 37774: week 65, Mmu 38560: week 67, and Mmu 38606: week 69). The dotted line indicates the lower limit of detection of the standard viral load assay (50 copies of viral RNA per mL of plasma).</p

LLV mutations and their abundance in the pre-suppression profile^a

a<p>Mutations are numbered beginning with the first nucleotide/amino acid of RT. The region encoding RT amino acids 65-210 was sequenced from LLV samples. Mutation abundance in the pre-suppression profile is expressed as the percentage of 454 sequence reads encoding the given mutation. Dashes (-) indicate that the specified mutation was not observed in sequence reads at a pre-determined 0.5% read threshold. LLV mutations that were not observed in the pre-suppression profile are underlined. LLV mutations associated with RT drug resistance are indicated by bold italics. ND, not determined: samples were either unavailable or not tested because viral loads were below the amplification sensitivity of the 454 sequencing assay. Nucleotide: (nt)</p>b<p>Number of LLV sequences containing a specific mutation (shown at left).</p>c<p>Sample was processed; however, sequence data was not generated at this position.</p>d<p>Sample analyzed by single-genome amplification (SGA) and not 454 sequencing. Five sequences were generated by SGA at week 10 PI for RM Mmu 37969.</p>e<p>The LLV predominant plasma clone (PPC) mutation in RM Mmu 37969 was characterized by the following ten linked RT nucleotide substitutions: C258T, C291T, A304C,T336C, G364A, A378G, A483G, A484T, T537C, and G612A. Nine of these mutations were linked on two individual sequences obtained at week 10 PI. G612A was only present in one of the week 10 PI sequences and is listed separately.</p

Sample viral load and number of LLV sequences/sample^a

a<p>The number of sequences generated in each plasma sample is shown to the left of the sample's viral load which is denoted in parentheses.</p>b<p>Viral load (VL) was determined by our standard viral load assay which has a sensitivity of 50 vRNA copies/mL.</p>c<p>Time points where some LLV sequences contained mutations which were not observed in the pre-suppression profile</p>d<p>Samples were obtained at necropsy and approximately 20 mL of plasma was analyzed to generate LLV sequences.</p>e<p>Viral load determined by an unmodified ultracentrifugation viral load assay (Deere <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-Deere2" target="_blank">[48]</a> using triplicate 10 µL vRNA samples. Sensitivity was 2 vRNA copies/mL.</p

Analysis of multiply spliced transcripts in lymphoid tissue reservoirs of rhesus macaques infected with RT-SHIV during HAART.

Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⁺ T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads &lt;50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load

Viral decay kinetics in the highly active antiretroviral therapy-treated rhesus macaque model of AIDS.

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2-58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans