The Kinetics of Human Immunodeficiency Virus Reverse Transcription Are Slower in Primary Human Macrophages Than in a Lymphoid Cell Line

doi:10.1006/viro.1994.1169

Virology

Volume 200, Issue 1, April 1994, Pages 114-120

https://doi.org/10.1006/viro.1994.1169 Get rights and content

Abstract

Reverse transcription is a critical event in the life of a retrovirus and a potential determinant of viral infectivity. The kinetics of "endogenous" reactions are relatively well defined but little is known about HIV reverse transcription during infection. In this report, we have estimated the rate and efficiency of HIV-1 reverse transcription in primary macrophages and H9 lymphoid cells using quantitative PCR to detect intermediate cDNA structures. DNA synthesis is completed 12-16 hr after infection of H9 cells, but requires more than 36 hr in Mφ, owing to slower rates of extension and template switching. Reverse transcription was inefficient in both cell types and in H9 cells the kinetics were sufficiently well resolved to estimate that only one in three transcripts initiated were extended to full-length viral DNA. Slower DNA synthesis largely accounts for the longer replicative cycle of HIV in macrophages. The rate of reverse transcription, which may depend upon levels of deoxynucleotide triphosphate substrates, is a potential factor in modulating the permissiveness of macrophage populations in vivo.

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Cited by (60)

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages
2023, Journal of Biological Chemistry
HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.
Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages
2020, Journal of Biological Chemistry
Citation Excerpt :
Unlike infected CD4+ T cells, which undergo rapid cell death, myeloid cells display long-living phenotype following HIV-1 infections (3–5). In addition to that, HIV-1 exhibits more rapid replication kinetics in activated CD4+ T cells compared with that of nondividing myeloid cells (6–8). We have previously reported that the extremely low dNTP concentration found in macrophages (20–40 nm) kinetically restricts HIV-1 reverse transcription, which generally utilizes cellular dNTPs, whereas HIV-1 replicates at higher rate within the higher cellular dNTP pool (1–5 μm) found in activated CD4+ T cells (9).
Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain– and HD domain–containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase–mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.
Mechanistic and kinetic differences between reverse transcriptases of Vpx coding and non-coding lentiviruses
2015, Journal of Biological Chemistry
Citation Excerpt :
Indeed, we reported that human primary monocyte-derived macrophages have 50–200 times lower dNTP concentrations (20–40 nm) than activated CD4+ T cells (2–4 μm) (7, 8). Importantly, although HIV-1 replication and viral production are robust in activated CD4+ T cells, its replication in non-dividing macrophages is kinetically delayed (9, 10). Our studies demonstrate that the extremely low dNTP level found in macrophages mechanistically contributes to the delayed replication kinetics of HIV-1 in macrophages and non-dividing cells.
Among lentiviruses, HIV Type 2 (HIV-2) and many simian immunodeficiency virus (SIV) strains replicate rapidly in non-dividing macrophages, whereas HIV Type 1 (HIV-1) replication in this cell type is kinetically delayed. The efficient replication capability of HIV-2/SIV in non-dividing cells is induced by a unique, virally encoded accessory protein, Vpx, which proteasomally degrades the host antiviral restriction factor, SAM domain- and HD domain-containing protein 1 (SAMHD1). SAMHD1 is a dNTPase and kinetically suppresses the reverse transcription step of HIV-1 in macrophages by hydrolyzing and depleting cellular dNTPs. In contrast, Vpx, which is encoded by HIV-2/SIV, kinetically accelerates reverse transcription by counteracting SAMHD1 and then elevating cellular dNTP concentration in non-dividing cells. Here, we conducted the pre-steady-state kinetic analysis of reverse transcriptases (RTs) from two Vpx non-coding and two Vpx coding lentiviruses. At all three sites of the template tested, the two RTs of the Vpx non-coding viruses (HIV-1) displayed higher k_pol values than the RTs of the Vpx coding HIV-2/SIV, whereas there was no significant difference in the K_d values of these two groups of RTs. When we employed viral RNA templates that induce RT pausing by their secondary structures, the HIV-1 RTs showed more efficient DNA synthesis through pause sites than the HIV-2/SIV RTs, particularly at low dNTP concentrations found in macrophages. This kinetic study suggests that RTs of the Vpx non-coding HIV-1 may have evolved to execute a faster k_pol step, which includes the conformational changes and incorporation chemistry, to counteract the limited dNTP concentration found in non-dividing cells and still promote efficient viral reverse transcription.
Restricted 5′-end gap repair of HIV-1 integration due to limited cellular dNTP concentrations in human primary macrophages
2013, Journal of Biological Chemistry
Citation Excerpt :
We transduced human primary MDMs with a vesicular stomatitis virus glycoprotein-pseudotyped pD3HIV-GFP vector, which encodes the entire HIV-1 genome except env and nef, which were replaced with enhanced GFP. Because reverse transcription in macrophages reaches completion between 24 and 72 h after initial infection (15, 39, 40), macrophages were initially transduced by HIV-1 vector for 48 h, and further reverse transcription and integration were blocked by treating the cells with 5 μm nevirapine (41) and 1 μm raltegravir, respectively (Fig. 6A). Note that these drugs do not interfere with the 5′-end gap repair machinery (42).
HIV-1 proviral DNA integration into host chromosomal DNA is only partially completed by the viral integrase, leaving two single-stranded DNA gaps with 5′-end mismatched viral DNA flaps. It has been inferred that these gaps are repaired by the cellular DNA repair machinery. Here, we investigated the efficiency of gap repair at integration sites in different HIV-1 target cell types. First, we found that the general gap repair machinery in macrophages was attenuated compared with that in dividing CD4⁺ T cells. In fact, the repair in macrophages was heavily reliant upon host DNA polymerase β (Pol β). Second, we tested whether the poor dNTP availability found in macrophages is responsible for the delayed HIV-1 proviral DNA integration in this cell type because the K_m value of Pol β is much higher than the dNTP concentrations found in macrophages. Indeed, with the use of a modified quantitative AluI PCR assay, we demonstrated that the elevation of cellular dNTP concentrations accelerated DNA gap repair in macrophages at HIV-1 proviral DNA integration sites. Finally, we found that human monocytes, which are resistant to HIV-1 infection, exhibited severely restricted gap repair capacity due not only to the very low levels of dNTPs detected but also to the significantly reduced expression of Pol β. Taken together, these results suggest that the low dNTP concentrations found in macrophages and monocytes can restrict the repair steps necessary for HIV-1 integration.
Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle
2013, Mucosal Immunology
Cervical tissue explants (CTEs) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTEs were productively infected in terms of HIV-1 p24_Gag release in culture supernatants, whereas 14 were not. Nonetheless, both accumulation of HIV-1_gag DNA and of p24_Gag⁺ CD4⁺ T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTEs. Nonproductive CTEs differed from productive CTEs for higher secretion of C-C motif chemokine ligand 3 (CCL3) and CCL5. A post-hoc analysis revealed that all productive CTEs were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTEs were derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle.
Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells
2023, Retrovirology

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Regular ArticleThe Kinetics of Human Immunodeficiency Virus Reverse Transcription Are Slower in Primary Human Macrophages Than in a Lymphoid Cell Line

Abstract

Regular Article
The Kinetics of Human Immunodeficiency Virus Reverse Transcription Are Slower in Primary Human Macrophages Than in a Lymphoid Cell Line