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Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy
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Significance
The presence of "defective" HIV-1 proviruses in HIV-infected patients has been well documented. The current consensus view of the "defective" proviruses is that these are dead-end products that do not give rise to progeny virus and thus collectively represent a "graveyard" of viruses. We describe the presence of defective HIV-1 proviruses capable of transcribing novel unspliced HIV-RNA species in HIV-infected patients on combination antiretroviral therapy. We propose that the proviruses persistently present in combination antiretroviral therapy-treated patients are not defective in a conventional sense, but rather represent incomplete forms of proviruses encoding translationally competent HIV-RNA transcripts. Strategies directed toward curing HIV-1 infection and eliminating the state of persistent immune activation need to include approaches designed to eliminate cells harboring such proviruses.
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Finding May Help Explain Persistent Immune Activation During Suppressive HIV Treatment
July 18, 2016
WHAT: Investigators from the National Institutes of Health have discovered that cells from HIV-infected people whose virus is suppressed with treatment harbor defective HIV DNA that can nevertheless be transcribed into a template for producing HIV-related proteins. This finding may affect scientists' understanding of the long-term effects of HIV infection and what a cure would require.
When HIV infects a cell, it inserts its genetic instructions into the cell's DNA.
Effective treatment with anti-HIV drugs does not eliminate this HIV DNA (called proviral DNA or a provirus), so in theory it could give rise to new viruses during treatment.
However, scientists previously have found that 95 percent or more of HIV proviruses are unable to encode intact viruses due to genetic mutations and deletions. As a result, researchers have come to think of these defective HIV proviruses as biological dead-ends.
This thinking may change thanks to the new finding by scientists in the Laboratory of Immunoregulation at the National Institute of Allergy and Infectious Diseases (NIAID), part of NIH.
Hiromi Imamichi, Ph.D., and colleagues used a technique for creating multiple copies of nearly full-length proviral DNA and cell-associated HIV RNA. The scientists showed that HIV RNAs complementary to defective proviruses could be found in cells from two of four people in whom treatment had suppressed the virus to undetectable levels for more than 8 years. This was evidence that the defective provirus had been transcribed from DNA into an RNA molecule. The researchers then demonstrated that these RNAs could encode novel HIV-related proteins. Thus, while unable to encode a virus, the defective proviral DNA could encode an intact protein.
This finding could help explain the persistent immune activation observed in people living with HIV who have undetectable levels of virus, say the study authors. The discovery also suggests another potential barrier to an HIV cure. More research is needed, however, to determine the impact of HIV RNA transcripts from defective proviruses, the authors add.
ARTICLE: H Imamichi et al. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy. PNAS DOI: 10.1073/pnas.1609057113 (2016).
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Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy
PNAS Aug 2016 - Hiromi Imamichia, Robin L. Dewarb, Joseph W. Adelsbergerb, Catherine A. Rehma, Una O'Dohertyc, Ellen E. Paxinosd, Anthony S. Faucia,1, and H. Clifford Lanea
aLaboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bClinical Services Program, Applied and Development Research Directorate, Leidos Biomedical Research, Inc., Frederick, MD 21072; cDepartment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and dApplications and Collaborations, Pacific Biosciences,
Menlo Park, CA 94025
Abstract
Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.
Discussion
In conclusion, cells harboring defective proviral DNA capable of transcribing novel usHIV-RNAs with translationally competent ORFs can be seen in HIV-infected patients even despite a period of prolonged viral suppression. In the present study, we were able to detect matching "defective" proviral DNAs and RNA transcripts in four instances in two separate patients. The presence of transcription-competent "defective" proviruses is a consistent finding in patients at all stages of HIV-1 infection. We have referred to these proviruses as "zombie" proviruses (34), as they are not really "alive" given that they lack the ability to produce intact viruses but can inflict harm by producing foreign nucleic acids and proteins. It is not clear what fraction of cells harboring defective proviruses produce RNA transcripts. It is possible that differences in the activation status of the host-infected cells or epigenetic state of the HIV-1 proviruses might play a role. Persistence of these proviruses may explain the persistent seropositivity to HIV-1 and persistent immune activation seen in patients with "undetectable" virus. In addition to eliciting persistent antibody responses, HIV-1 proteins derived from "defective" proviruses could also be associated with CD4 and CD8 T-cell responses. Future studies will hopefully further delineate the effects of the transcription or translation of defective proviruses on immune responses and immune activation in cART-treated HIV-infected individuals with prolonged viral suppression. Strategies directed toward curing HIV-1 infection and eliminating the state of persistent immune activation associated with HIV-1 infection need to include approaches designed to eliminate cells harboring such proviruses.
Although once considered a terminal illness, HIV-1 infection has now become a chronic manageable disease. With the use of combination antiretroviral therapy (cART), prolonged viral suppression of plasma HIV-RNA levels (pVL) < 40 copies per milliliter is achievable in the majority of patients with HIV-1 infection (1). One paradoxical observation is that, despite prolonged viral suppression with no evidence of active viral replication, the majority of HIV-infected patients exhibit persistent presence of HIV-1 proviruses (2↓-4), persistent seropositivity to HIV-1 (5), and evidence of immune activation (6↓↓↓-10). The only setting in which this has not been the case has been following bone marrow transplantation in which seronegativity has been reported in association with a loss of peripheral blood proviral DNA (11, 12). Over 90% of proviruses in the peripheral blood are thought to be "defective" by having lethal genetic alterations that include G-to-A hypermutations (13, 14) and small insertions/deletions (indels) that disrupt ORFs (13), or large internal deletions (13, 15, 16). Because these defective proviruses are unable to encode intact viruses, the peripheral blood pool of proviruses has been thought to largely represent a silent "graveyard" of viral sequences. In a single case, we previously reported the ability of a provirus with a stop codon in the protease to transcribe viral RNA (14). Cells harboring these "defective" proviruses were found in a clonally expanded population of effector memory CD4+T cells that had persisted for 17 y. To better evaluate the characteristics of these persistent proviruses, we developed a system that allows simultaneous isolation of genomic DNA and cytoplasmic RNA from the same populations of CD4+ T cells, amplification of up to 8.9 kb HIV-1 DNA and 8.4 kb cell-associated unspliced HIV-RNA (usHIV-RNA), and sequencing of the resulting near full-length HIV-1 genome fragments. This approach allowed us to better characterize the genetic variability in HIV-1 proviral genomes and to determine which "defective" proviruses were transcribed and capable of encoding viral proteins.
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