NIH Scientists Discover that Defective HIV DNA Can
Encode HIV-Related Proteins, in patients on cART
Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy
PNAS July 18 2016 published ahead of print
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
Contributed by Anthony S. Fauci, June 9, 2016 (sent for review April 23, 2016; reviewed by Scott Hammer and Nelson L. Michael)
"....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."
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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.
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.
For Immediate Release: Monday, July 18, 2016
NIH Scientists Discover that Defective HIV DNA Can Encode HIV-Related Proteins
Finding May Help Explain Persistent Immune Activation During Suppressive HIV Treatment
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.
H Imamichi et al. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy. PNAS DOI:
NIAID Director Anthony S. Fauci, M.D., and H. Clifford Lane, M.D., director of the NIAID Division of Clinical Research, are available for interviews. Dr. Fauci is chief of the NIAID Laboratory of Immunoregulation, and Dr. Lane is chief of the Clinical and Molecular Retrovirology Section within the laboratory.
To schedule interviews, please contact Laura S. Leifman, (301) 402-1663, firstname.lastname@example.org.
NIAID conducts and supports research—at NIH, throughout the United States, and worldwide—to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID website.
About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.
Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy
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&8659;-4), persistent seropositivity to HIV-1 (5), and evidence of immune activation (6&8659;&8659;&8659;-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.
Characterization of HIV-1 Proviruses in Patients with HIV-1 Infection.
The 5'LTR-to-3'LTR single genome amplification and direct amplicon sequencing of HIV-1 proviruses were performed for four patients with pVL ≥ 40 copies per milliliter (range 26,758-225,658); four patients with pVL < 40 copies per milliliter for >6 y (range 6.1-11.9 y); and one patient before and after suppressive cART. Proviral DNA lengths were estimated based on migration distances of the 5'LTR-to-3'LTR PCR-amplified DNA fragments on agarose gels and confirmed by sequencing (Fig. 1 A-C). For the four patients with pVL ≥ 40 copies per milliliter, 42 of 48 (88%) full-length proviral genomes encoded for an intact virus. In contrast, for the four patients with pVL < 40 copies per milliliter, full-length genomes were found in only one patient [patient (Pt) 8]. In that patient, six of the seven sequenced proviruses were defective with lethal mutations in their genomes (Fig. 1B). The higher proportion of full-length proviruses observed in patients with pVL ≥ 40 copies per milliliter likely reflects the presence of substantial ongoing viral replication that was taking place in these patients at the time of sampling. Of note, such truncated proviruses were not detected when either a plasmid DNA containing the full-length NL4.3 strain of HIV-1 or genomic DNA from the chronically infected 8E5 cell line were used as PCR templates (Fig. S1), thus minimizing the possibility that these findings were a result of PCR artifacts. However, these truncated proviruses could be detected as early as 24 h following in vitro infection of primary CD4+ T cells with the DH12 clone of HIV-1 (Fig. S2).
The presence of clonally expanded defective provirus populations was evident in all four patients with pVL < 40 copies per milliliter (Fig. 1B, clones i-vii). Of particular note, the 3.0-kb clone ii proviruses (Fig. 1B) accounted for 56% (15 of 27) of the total number of proviruses found in patient 5. Clonally expanded proviruses were not detected in any of the four patients with pVL ≥ 40 copies per milliliter. We believe this dichotomy likely reflects the relative proportions of intact and truncated proviruses in these two subpopulations of patients.
To directly determine the effects of therapy and to evaluate changes in the distribution of HIV-1 proviral DNA lengths over time, samples from a single patient (patient 9) from 1995 and 2001 were examined (Fig. 1 D-F). Before initiation of cART and in the setting of a higher viral load (pVL = 89,449 copies per milliliter), the frequency of full-length proviruses was 36% (14 of 39). Six years later and 3 y after achieving pVL < 50 copies per milliliter, this percentage had decreased to 6% (2 of 33). This finding is consistent with the cross-sectional data presented in Fig. 1 A and B in that the frequencies of full-length proviruses were higher when there was evidence of active viral replication.
Deletion Junction Analysis of HIV-1 Proviruses.
Among the truncated proviruses derived from the nine patients, deletion sizes ranging from 251 bp to 8,033 bp in length were identified (Fig. S3A). Approximately 40% of deletions were associated with directly repeated sequences at the deletion junctions, suggesting that they had occurred during the negative-strand synthesis phase of reverse transcription (Fig. S3) (15, 17, 18). In one instance in patient 5 and two instances in patient 9 (Fig. S3A), these deletions included the 15-nt stretch of purine-rich sequence referred to as the polypurine tract (PPT) that is located in the pol gene (cPPT) and the nef/U3 region of HIV-1 (3'PPT) (19). The mechanisms responsible for the remaining 60% of deletions are unknown. Of note, in patient 9, we identified a provirus with a "deletion-insertion-deletion" with insertion of host sequence B4GALT5 (Fig. 1 E and F). "Deletion-inversion-deletion" complex genome structures were found in patient 2 (Fig. 1A and Fig. S3A), patient 9 (Fig. 1 E and F), and patient 5 (Fig. 1B and Fig. S3A). Sequence analyses of these proviruses indicated that incorporation of the host sequence by readthrough of HIV-1 polyadenylation signal transcription (20&8659;-22) or a single polymerase jump during reverse transcription (15, 17, 18) were unlikely mechanisms to explain the unique "insertion or inversion in deletions" genome structures we observed. Taken together, these findings highlight the fact that multiple mechanisms may be responsible for alterations in the HIV-1 proviral genome. As noted below, some of these alterations may lead to unique ORFs.
As seen in Fig. S3A, many of the sequenced truncated proviruses contained, at a minimum, a short stretch of the 5' end of the gag-pol intronic region and a part of the Nef coding region, although lacking the second intronic region encoding most of the accessory proteins and Env (Fig. S4). If transcription occurred from any of these truncated proviruses, the transcripts would resemble unspliced (genomic) HIV-RNA with removal of the second intronic region.
Characterization of Novel usHIV-1 RNA Species.
To determine whether or not any of these "defective" proviruses led to the production of novel usHIV-RNA that could lead to the production of novel proteins in the setting of prolonged viral suppression, we performed limiting-dilution single-genome amplification and directly sequenced the resulting single-genome amplicons. Nested PCR reactions were performed with two forward PCR primers located immediately downstream of the major 5' splice donor site (D1) within the gag-pol intronic region (Fig. S4). These custom-designed PCR primers enabled us to selectively amplify unspliced forms of HIV-RNA species of up to 8.4 kb in size from patient 1 (pVL = 225,638 copies per milliliter) and patients 5-8 (pVL < 40 copies per milliliter). The distribution of cell-associated usHIV-RNA lengths are shown in Fig. 2A and plotted alongside the length distributions of the proviral DNA genomes derived from the same populations of CD4+ T cells.
For the patient with clear evidence of substantial active virus replication (Pt 1 in Fig. 2A and Fig. S5), 12 of 25 (48%) of HIV-1 proviruses were full-length, as were 8 of 11 (73%) of the cell-associated usHIV-RNA transcripts. In contrast, for the four patients (Pts 5-8) with pVL < 40 copies per milliliter, full-length proviruses were found in only one patient (Pt 8) and full-length usHIV-RNA species were not detected in any of these patients (Fig. 2A and Fig. S5).
The in vivo presence of clonally expanded proviral populations has been reported by ourselves and others (14, 23&8659;&8659;-26). However, there is minimal evidence for transcription of defective proviruses (14, 27). Here, we were able to identify the presence of clonally expanded defective provirus populations and to demonstrate ongoing transcription of these expanded "defective" proviral clones in two different patients (Pts 5 and 7) with pVL < 40 copies per milliliter (clones ii and vi in Fig. 2A and Fig. S5). In two additional instances (pairs "a" and "b") in the same two patients (Pts 5 and 7), we were able to identify proviral DNA-usHIV-RNA pairs, providing strong evidence that, at best, some of these "defective" proviruses were responsible for the production of novel usHIV-RNA species in patients with "undetectable" plasma viremia (Fig. 2A and Fig. S5).
Sequence analyses of these novel usHIV-RNA transcripts revealed exon structures that were very different from those of the known spliced HIV-1 mRNA variants and they ranged in size from 0.5 kb to 2.6 kb (Fig. 2B). These novel usHIV-RNA transcripts had appropriate transcription start and stop signals and thus possessed translationally competent ORFs. At a minimum, a part of the gag and nef genes were retained in all of these truncated novel usHIV-RNA transcripts. Some also included elements of pol, env, and rev to encode a series of novel HIV-1 chimeric proteins (Fig. 2C).
As noted in Fig. 2B, the novel usHIV-RNA transcripts reported in this study did not contain any of the classic splice acceptor/donor sites in their genomes (28), nor did they contain cryptic splice sites that have been reported elsewhere (29, 30). These data support the hypothesis that the "defective" proviruses seen in patients with HIV-1 infection lead to the production of novel HIV-RNA transcripts faithfully transcribed from these proviruses.
All of the usHIV-RNA transcripts found in patients 5-8 were missing a gene segment encoding the Rev response element (RRE) (Fig. 2B). Two usHIV-RNA transcripts contained a partial rev gene without the RRE (2.0 kb in Pt 5 and 1.3 kb in Pt 6). In general, HIV-RNAs without any intact introns (i.e., fully spliced 2-kb HIV-RNA transcripts) or cis-acting repressive sequences (CRS) elements can be exported to the cytoplasm independent of Rev/RRE via the host cell TAP-dependent mRNA export pathway that mediates general mRNA export (28, 31) and are able to productively engage the host cell's translational machinery. In fact, a partial removal of the CRS elements (the ones within pol) has led to Rev/RRE-independent nuclear export of the transcripts and protein production, albeit at low levels in in vitro systems (32, 33). The fact that novel usHIV-RNAs reported in the present study do not contain any intact intronic regions (defined by splice donor/acceptor sites: D1-A1 and D2-A7) (Fig. 2B) or the CRS element within pol and thus are of "intron-free" structures might explain the Rev/RRE-independent nuclear export of these variants.
Assessment of the Pattern of HIV-1 Protein Production in Vivo.
Taken together, these data support our hypothesis that patients with HIV-1 infection, despite plasma pVL < 40 copies per milliliter, may still be capable of producing viral proteins. This finding may help explain the persistence of antibodies to HIV-1 proteins greater than 10 y following initiation of suppressive cART (Fig. 3). Of note, the stochastic element to the fading of bands suggest an asynchronous decay in the viral proteins and is consistent with the production and decay of novel chimeric proteins, which can be seen by comparing the persistence of antibodies to Env in patient 5 and the relative persistence of antibodies to Gag in patient 6 with the uniform presence of antibodies to all viral proteins in patients 1-4.
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.