HIV-1 Capsid Protein Research Articles

A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

Alternate conformational states of the HIV-1 capsid protein: Atomistic structures and dynamics of interconversion.

The HIV-1 capsid protein assembles into a conical shell that encases the viral RNA genome. The capsid protein is a two-domain protein, with the C-terminal domain (CTD) forming a dimer. Depending on being in a solution environment or the crystalline state, different dimer arrangements have been observed for the full capsid protein, as well as a truncated construct consisting of only the CTD. To characterize the structures and dynamics of these alternate quaternary arrangements, we carried out atomistic simulations using the weighted ensemble enhanced sampling strategy. Our simulations generated continuous pathways for interconversion between the two states with rate constants that agree with those measured by 19F NMR exchange spectroscopy. Our results demonstrate the advantages of pairing atomistic simulations with 19F NMR and have implications for the structural polymorphism of the HIV-1 viral capsid assembly process.

Critical mechanistic features of HIV-1 viral capsid assembly

The maturation of HIV-1 capsid protein (CA) into a cone-shaped lattice capsid is critical for viral infectivity. CA can self-assemble into a range of capsid morphologies made of ~175 to 250 hexamers and 12 pentamers. The cellular polyanion inositol hexakisphosphate (IP6) has recently been demonstrated to facilitate conical capsid formation by coordinating a ring of arginine residues within the central cavity of capsid hexamers and pentamers. However, the kinetic interplay of events during IP6 and CA coassembly is unclear. In this work, we use coarse-grained molecular dynamics simulations to elucidate the molecular mechanism of capsid formation, including the role played by IP6. We show that IP6, in small quantities at first, promotes curvature generation by trapping pentameric defects in the growing lattice and shifts assembly behavior toward kinetically favored outcomes. Our analysis also suggests that IP6 can stabilize metastable capsid intermediates and can induce structural pleomorphism in mature capsids.

Low-molecular-weight anti-HIV-1 agents targeting HIV-1 capsid proteins.

The HIV-1 capsid is a shell that encapsulates viral RNA, and forms a conical structure by assembling oligomers of capsid (CA) proteins. Since the CA proteins are highly conserved among many strains of HIV-1, the inhibition of the CA function could be an appropriate goal for suppression of HIV-1 replication, but to date, no drug targeting CA has been developed. Hydrophobic interactions between two CA molecules through Trp184 and Met185 in the protein are known to be indispensable for conformational stabilization of the CA multimer. In our previous study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site was synthesized and found to have significant anti-HIV-1 activity. In the present study, molecules with different scaffolds based on a dipeptide mimic of Trp184 and Met185 have been designed and synthesized. Their significant anti-HIV activity and their advantages compared to the previous compounds were examined. The present results should be useful in the design of novel CA-targeting anti-HIV agents.

The discovery and design of novel HIV-1 capsid modulators and future perspectives

ABSTRACT Introduction Although combination antiretroviral therapy (cART) has achieved significant success in treating HIV, the emergence of multidrug-resistant viruses and cumulative medication toxicity make it necessary to find new classes of antiretroviral agents with novel mechanisms of action. With high sequence conservation, the HIV-1 capsid (CA) protein has attracted attention as a prospective therapeutic target due to its crucial structural and regulatory functions in the HIV-1 replication cycle. Area covered Herein, the authors provide a cutting-edge overview of current advances in the design and discovery of CA modulators, PF74, GS-6207 and their derivativeswhich targets a therapeutically attractive NTD-CTD interprotomer pocket within the hexameric configuration of HIV-1 CA. The discovery and development of these compounds, and derivatives thereof, have provided valuable information for the design of second-generation CA-targeting antivirals. Expert opinion Despite some successes in designing and discovering HIV-1 CA modulators, more studies are required to decipher which chemical groups confer specific desirable properties. The future of CA-modulating compounds may lie in covalent inhibition and the creation of proteolysis-targeting chimeras (PROTACs). Moreover, biological interrogation of the process of CA uncoating, virus–host interactions, and studies on the lattice-binding restriction factors may improve our knowledge of HIV-1 CA and support the design of new antiviral agents.

Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction

Protein-protein interactions are fundamental to life processes. Complementary computational, structural, and biophysical studies of these interactions enable the forces behind their specificity and strength to be understood. Antibody fragments such as single-chain antibodies have the specificity and affinity of full antibodies but a fraction of their size, expediting whole molecule studies and distal effects without exceeding the computational capacity of modeling systems. We previously reported the crystal structure of a high-affinity nanobody 59H10 bound to HIV-1 capsid protein p24 and deduced key interactions using all-atom molecular dynamics simulations. We studied the properties of closely related medium (37E7) and low (48G11) affinity nanobodies, to understand how changes of three (37E7) or one (48G11) amino acids impacted these interactions; however, the contributions of enthalpy and entropy were not quantified. Here, we report the use of qualitative and quantitative experimental and in silico approaches to separate the contributions of enthalpy and entropy. We used complementary circular dichroism spectroscopy and molecular dynamics simulations to qualitatively delineate changes between nanobodies in isolation and complexed with p24. Using quantitative techniques such as isothermal titration calorimetry alongside WaterMap and Free Energy Perturbation protocols, we found the difference between high (59H10) and medium (37E7) affinity nanobodies on binding to HIV-1 p24 is entropically driven, accounted for by the release of unstable waters from the hydrophobic surface of 59H10. Our results provide an exemplar of the utility of parallel in vitro and in silico studies and highlight that differences in entropic interactions between amino acids and water molecules are sufficient to drive orders of magnitude differences in affinity.

Discovery and Mechanistic Investigation of Piperazinone Phenylalanine Derivatives with Terminal Indole or Benzene Ring as Novel HIV-1 Capsid Modulators.

HIV-1 capsid (CA) performs multiple roles in the viral life cycle and is a promising target for antiviral development. In this work, we describe the design, synthesis, assessment of antiviral activity, and mechanistic investigation of 20 piperazinone phenylalanine derivatives with a terminal indole or benzene ring. Among them, F2-7f exhibited moderate anti-HIV-1 activity with an EC50 value of 5.89 μM, which was slightly weaker than the lead compound PF74 (EC50 = 0.75 μM). Interestingly, several compounds showed a preference for HIV-2 inhibitory activity, represented by 7f with an HIV-2 EC50 value of 4.52 μM and nearly 5-fold increased potency over anti-HIV-1 (EC50 = 21.81 μM), equivalent to PF74 (EC50 = 4.16 μM). Furthermore, F2-7f preferred to bind to the CA hexamer rather than to the monomer, similar to PF74, according to surface plasmon resonance results. Molecular dynamics simulation indicated that F2-7f and PF74 bound at the same site. Additionally, we computationally analyzed the ADMET properties for 7f and F2-7f. Based on this analysis, 7f and F2-7f were predicted to have improved drug-like properties and metabolic stability over PF74, and no toxicities were predicted based on the chemotype of 7f and F2-7f. Finally, the experimental metabolic stability results of F2-7f in human liver microsomes and human plasma moderately correlated with our computational prediction. Our findings show that F2-7f is a promising small molecule targeting the HIV-1 CA protein with considerable development potential.

Design, Synthesis, and Mechanistic Study of 2-Pyridone-Bearing Phenylalanine Derivatives as Novel HIV Capsid Modulators.

The AIDS pandemic is still of importance. HIV-1 and HIV-2 are the causative agents of this pandemic, and in the absence of a viable vaccine, drugs are continually required to provide quality of life for infected patients. The HIV capsid (CA) protein performs critical functions in the life cycle of HIV-1 and HIV-2, is broadly conserved across major strains and subtypes, and is underexploited. Therefore, it has become a therapeutic target of interest. Here, we report a novel series of 2-pyridone-bearing phenylalanine derivatives as HIV capsid modulators. Compound FTC-2 is the most potent anti-HIV-1 compound in the new series of compounds, with acceptable cytotoxicity in MT-4 cells (selectivity index HIV-1 > 49.57; HIV-2 > 17.08). However, compound TD-1a has the lowest EC50 in the anti-HIV-2 assays (EC50 = 4.86 ± 1.71 μM; CC50= 86.54 ± 29.24 μM). A water solubility test found that TD-1a showed a moderately increased water solubility compared with PF74, while the water solubility of FTC-2 was improved hundreds of times. Furthermore, we use molecular simulation studies to provide insight into the molecular contacts between the new compounds and HIV CA. We also computationally predict drug-like properties and metabolic stability for FTC-2 and TD-1a. Based on this analysis, TD-1a is predicted to have improved drug-like properties and metabolic stability over PF74. This study increases the repertoire of CA modulators and has important implications for developing anti-HIV agents with novel mechanisms, especially those that inhibit the often overlooked HIV-2.

Design, Synthesis and Structure-Activity Relationships of Phenylalanine-Containing Peptidomimetics as Novel HIV-1 Capsid Binders Based on Ugi Four-Component Reaction.

As a key structural protein, HIV capsid (CA) protein plays multiple roles in the HIV life cycle, and is considered a promising target for anti-HIV treatment. Based on the structural information of CA modulator PF-74 bound to HIV-1 CA hexamer, 18 novel phenylalanine derivatives were synthesized via the Ugi four-component reaction. In vitro anti-HIV activity assays showed that most compounds exhibited low-micromolar-inhibitory potency against HIV. Among them, compound I-19 exhibited the best anti-HIV-1 activity (EC50 = 2.53 ± 0.84 μM, CC50 = 107.61 ± 27.43 μM). In addition, I-14 displayed excellent HIV-2 inhibitory activity (EC50 = 2.30 ± 0.11 μM, CC50 > 189.32 μM) with relatively low cytotoxicity, being more potent than that of the approved drug nevirapine (EC50 > 15.02 μM, CC50 > 15.2 μM). Additionally, surface plasmon resonance (SPR) binding assays demonstrated direct binding to the HIV CA protein. Moreover, molecular docking and molecular dynamics simulations provided additional information on the binding mode of I-19 to HIV-1 CA. In summary, we further explored the structure—activity relationships (SARs) and selectivity of anti-HIV-1/HIV-2 of PF-74 derivatives, which is conducive to discovering efficient anti-HIV drugs.

Metabolite Identification of HIV-1 Capsid Modulators PF74 and 11L in Human Liver Microsomes.

PF74 and 11L, as potent modulators of the HIV-1 capsid protein, have been demonstrated to act at both early and late stages in the HIV-1 life cycle. However, their clearance is high in human liver microsomes (HLMs). The main goal of this study was to clarify the metabolism of PF74 and 11L in HLMs, and provide guidance for future structural optimization. To accomplish this, the phase-I metabolites of PF74 and 11L, resulting from in vitro incubation with HLMs, were investigated via ultra-performance liquid chromatography–ultraviolet–high-resolution mass spectrometry (UPLC–UV–HRMS). The results show that 17 phase-I metabolites were putatively annotated for PF74, whereas 16 phase-I metabolites were found for 11L. The main metabolic pathways of PF74 in HLMs were oxidation and demethylation, and the secondary metabolic pathway was hydrolysis; thus, the di-oxidation and demethylation products (M7, M9, M11, and M14) were found to be major metabolites of PF74 in HLMs. In comparison, the main metabolic pathways of 11L in HLMs were oxidation, demethylation, dehydrogenation, and oxidative deamination, with M6′, M11′, M15′, and M16′ as the main metabolites. We suggest that the indole ring and N-methyl group of PF74, and the aniline group, benzene ring R1′, N-methyl, and methoxy group of 11L, were the main metabolic soft spots. Therefore, our research illuminates structural optimization options in seeking improved HIV-1 CA modulators.

From design to biological mechanism evaluation of phenylalanine-bearing HIV-1 capsid inhibitors targeting a vital assembly interface

HIV-1 capsid protein (CA) has emerged as a promising target for antiviral treatment considering its structural and regulatory roles in HIV-1 replication. Here, we disclose the design, synthesis, biological assessment, and mechanism investigation of a novel series of phenylalanine derivatives gained by further structural modification of PF74. The newly synthesized compounds demonstrated potent anti-HIV activity, represented by 7n displayed anti-HIV-1 activity 6.25-fold better than PF74, and 7h showed anti-HIV-2 activity with nearly 139 times improved efficacy over PF74. Surface plasmon resonance (SPR) studies of representative compounds proved that HIV-1 CA was the binding target. Competitive SPR studies using CPSF6 and NUP153 peptides identified that 7n binds to a vital CA assembly interface between the N-terminal and C-terminal domain (NTD-CTD interface). Action stage determination assay revealed that the newly synthesized compounds were antiviral with a dual-stage inhibitory profile. Molecular dynamics (MD) simulations offered the crucial foundation for the hopeful antiviral potency of 7n. Besides, 7m and 7n modestly increased metabolic stabilities in human liver microsome (HLM) and human plasma compared to PF74. Overall, these studies offer valuable insights and can regard as the beginning for succedent medicinal chemistry endeavors to discover promising HIV capsid inhibitors with improved efficacy and better drug-like characteristics.

Clade-Specific Alterations within the HIV-1 Capsid Protein with Implications for Nuclear Translocation.

The HIV-1 capsid (CA) protein has emerged as an attractive therapeutic target. However, all inhibitor designs and structural analyses for this essential HIV-1 protein have focused on the clade B HIV-1 (NL4-3) variant. This study creates, overproduces, purifies, and characterizes the CA proteins from clade A1, A2, B, C, and D isolates. These new CA constructs represent novel reagents that can be used in future CA-targeted inhibitor design and to investigate CA proteins’ structural and biochemical properties from genetically diverse HIV-1 subtypes. Moreover, we used surface plasmon resonance (SPR) spectrometry and computational modeling to examine inter-clade differences in CA assembly and binding of PF-74, CPSF-6, and NUP-153. Interestingly, we found that HIV-1 CA from clade A1 does not bind to NUP-153, suggesting that the import of CA core structures through the nuclear pore complex may be altered for viruses from this clade. Overall, we have demonstrated that in silico generated models of the HIV-1 CA protein from clades other than the prototypically used clade B have utility in understanding and predicting biology and antiviral drug design and mechanism of action.

Sub-picomolar lateral flow antigen detection with two-wavelength imaging of composite nanoparticles

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0–5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Structure-based virtual screening workflow to identify antivirals targeting HIV-1 capsid.

We have identified novel HIV-1 capsid inhibitors targeting the PF74 binding site. Acting as the building block of the HIV-1 capsid core, the HIV-1 capsid protein plays an important role in the viral life cycle and is an attractive target for antiviral development. A structure-based virtual screening workflow for hit identification was employed, which includes docking 1.6 million commercially-available drug-like compounds from the ZINC database to the capsid dimer, followed by applying two absolute binding free energy (ABFE) filters on the 500 top-ranked molecules from docking. The first employs the Binding Energy Distribution Analysis Method (BEDAM) in implicit solvent. The top-ranked compounds are then refined using the Double Decoupling method in explicit solvent. Both docking and BEDAM refinement were carried out on the IBM World Community Grid as part of the FightAIDS@Home project. Using this virtual screening workflow, we identified 24 molecules with calculated binding free energies between − 6 and − 12 kcal/mol. We performed thermal shift assays on these molecules to examine their potential effects on the stability of HIV-1 capsid hexamer and found that two compounds, ZINC520357473 and ZINC4119064 increased the melting point of the latter by 14.8 °C and 33 °C, respectively. These results support the conclusion that the two ZINC compounds are primary hits targeting the capsid dimer interface. Our simulations also suggest that the two hit molecules may bind at the capsid dimer interface by occupying a new sub-pocket that has not been exploited by existing CA inhibitors. The possible causes for why other top-scored compounds suggested by ABFE filters failed to show measurable activity are discussed.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10822-022-00446-5.

Spatial and Genomic Correlates of HIV-1 Integration Site Targeting.

HIV-1 integrase and capsid proteins interact with host proteins to direct preintegration complexes to active transcription units within gene-dense regions of chromosomes for viral DNA integration. Analyses of spatially-derived genomic DNA coordinates, such as nuclear speckle-associated domains, lamina-associated domains, super enhancers, and Spatial Position Inference of the Nuclear (SPIN) genome states, have further informed the mechanisms of HIV-1 integration targeting. Critically, however, these different types of genomic coordinates have not been systematically analyzed to synthesize a concise description of the regions of chromatin that HIV-1 prefers for integration. To address this informational gap, we have extensively correlated genomic DNA coordinates of HIV-1 integration targeting preferences. We demonstrate that nuclear speckle-associated and speckle-proximal chromatin are highly predictive markers of integration and that these regions account for known HIV biases for gene-dense regions, highly transcribed genes, as well as the mid-regions of gene bodies. In contrast to a prior report that intronless genes were poorly targeted for integration, we find that intronless genes in proximity to nuclear speckles are more highly targeted than are spatially-matched intron-containing genes. Our results additionally highlight the contributions of capsid and integrase interactions with respective CPSF6 and LEDGF/p75 host factors in these HIV-1 integration targeting preferences.

Large scale all-atom molecular dynamics simulations of mutant CA tubes provide insights on cytotoxic T-lymphocyte-mediated HIV-1 restriction

For certain HIV-1-infected individuals, viral replication is controlled via expression of human leukocyte antigens (HLA). HLAs mark infected cells for host-mediated elimination by exposing viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). For HLA-B27, CTLs target and attack the HIV-1 capsid protein (CA), particularly the CA-derived KK10 epitope. Substitution mutation of Arginine at position 264 to Lysine (R264K) enables HIV-1 to escape the host CTL response but limits infectivity. Through a multi-pronged approach, employing both experimental and computational methods, we show that impairment of R264K mutant infectivity results from stymied viral DNA integration.

Uracil derivatives as HIV-1 capsid protein inhibitors: design, in silico, in vitro and cytotoxicity studies.

A series of novel uracil derivatives such as bispyrimidine dione and tetrapyrimidine dione derivatives were designed based on the existing four-point pharmacophore model as effective HIV capsid protein inhibitors. The compounds were initially docked with an HIV capsid protein monomer to rationalize the ideas of design and to find the potential binding modes. The successful design and computational studies led to the synthesis of bispyrimidine dione and tetrapyrimidine dione derivatives from uracil and aromatic aldehydes in the presence of HCl using novel methodology. The in vitro evaluation in HIV p24 assay revealed five potential uracil derivatives with IC50 values ranging from 191.5 μg ml−1 to 62.5 μg ml−1. The meta-chloro substituted uracil compound 9a showed promising activity with an IC50 value of 62.5 μg ml−1 which is well correlated with the computational studies. As expected, all the active compounds were noncytotoxic in BA/F3 and Mo7e cell lines highlighting the thoughtful design. The structure activity relationship indicates the position priority and lower log P values as the possible cause of inhibitory potential of the uracil compounds.

Lenacapavir: a first-in-class HIV-1 capsid inhibitor.

This review summarizes available data for lenacapavir, an investigational first-in-class agent that disrupts functioning of HIV capsid protein across multiple steps in the viral life cycle. Lenacapavir demonstrated picomolar potency in vitro with no cross resistance to existing antiretroviral classes and potent antiviral activity in persons with HIV-1. In persons with HIV-1, there was no preexisting resistance to lenacapavir regardless of treatment history. Lenacapavir can be administered orally either daily or weekly and subcutaneously up to every 6 months. In heavily treatment-experienced persons with multidrug-resistant HIV-1 and in treatment-naive persons with HIV-1, lenacapavir in combination with other antiretroviral agents led to high rates of virologic suppression and was well tolerated. Ongoing studies are evaluating long-acting dosing of lenacapavir for treating HIV-1 in combination with other antiretrovirals and preventing HIV-1 as a single agent.

Essential role of TOSO/FAIM3 in intestinal IgM reverse transcytosis.

Secretory immunoglobulin A (SIgA) can travel to and from the lumen and transport antigen to subepithelial cells. However, IgM can also multimerize into functional secretory component-bound immunoglobulin. While it is already known that both SIgA and SIgM undergo transcytosis to be secreted at the mucosal surface, only SIgA has been shown to perform retrotranscytosis through microfold cells (M cells) of the Peyer's patch. Here, we investigate whether SIgM could also be taken up by M cells via retrotranscytosis. This transport involves FcμR binding at the apical membrane of M cells. We then demonstrate that SIgM can be exploited by SIgM-p24 (HIV-capsid protein) complexes during immunization in the nasal- or gut-associated lymphoid tissue (NALT or GALT), conferring efficient immune responses against p24. Our data demonstrate a mucosal function of SIgM, which could play a role in the regulation of mucosal immunity.

Cooperative methylation of human tRNA3Lys at positions A58 and U54 drives the early and late steps of HIV-1 replication.

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N1-methyladenosine modification at tRNA position 58 (m1A58); however, the role of m1A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m1A in HIV-1 infection. We confirmed that tRNA3Lys m1A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m1A58-deficient tRNA3Lys instead of m1A58-modified tRNA3Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2′-O-dimethyluridine at U54 of tRNA3Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNALys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m1A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.