scholarly journals C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Fu-Hsien Yu ◽  
Kuo-Jung Huang ◽  
Chin-Tien Wang
Keyword(s):  
Leucine Zipper ◽  
Virus Assembly ◽  
Virus Production ◽  
Supporting Evidence ◽  
Dependent Manner ◽  
Dimer Formation ◽  
Reading Frame ◽  
Virus Maturation ◽  
C Terminus ◽  
Hiv 1

ABSTRACT HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.

scholarly journals Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Wenwen Zheng ◽  
Limian Ling ◽  
Zhaolong Li ◽  
Hong Wang ◽  
Yajuan Rui ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Virus Assembly ◽  
Virus Production ◽  
Bovine Immunodeficiency Virus ◽  
Reading Frame ◽  
Virion Infectivity Factor ◽  
Immunodeficiency Virus ◽  
New Strategy ◽  
Species Specific ◽  
Hiv 1

ABSTRACT The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies. IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein

2017 ◽  
Vol 474 (10) ◽  
pp. 1653-1668 ◽  
Author(s):  
Amjad Ali ◽  
Rameez Raja ◽  
Sabihur Rahman Farooqui ◽  
Shaista Ahmad ◽  
Akhil C. Banerjea
Keyword(s):  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Virus Production ◽  
Dependent Manner ◽  
Tat Protein ◽  
Viral Production ◽  
Cellular Processes ◽  
Latently Infected ◽  
Dub Inhibitor ◽  
Hiv 1

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

scholarly journals Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

2020 ◽  
Vol 295 (37) ◽  
pp. 13023-13030
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Minoru Tobiume ◽  
Satoshi Kishigami ◽  
Kenzo Tokunaga
Keyword(s):  
Virus Production ◽  
Integrase Inhibitor ◽  
Luciferase Assay ◽  
Protein Levels ◽  
C Terminus ◽  
P24 Protein ◽  
Luminescent Signal ◽  
Routine Handling ◽  
Hiv 1 ◽  
Peptide Tag

In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.

scholarly journals Important Role for the CA-NC Spacer Region in the Assembly of Bovine Immunodeficiency Virus Gag Protein

2004 ◽  
Vol 78 (2) ◽  
pp. 551-560 ◽  
Author(s):  
Xiaofeng Guo ◽  
Jing Hu ◽  
James B. Whitney ◽  
Rodney S. Russell ◽  
Chen Liang
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Bovine Immunodeficiency Virus ◽  
Gag Protein ◽  
Gag Proteins ◽  
Visna Virus ◽  
Immunodeficiency Virus ◽  
Anemia Virus ◽  
Hiv 1

ABSTRACT Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.

scholarly journals Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

2010 ◽  
Vol 84 (18) ◽  
pp. 9035-9046 ◽  
Author(s):  
Mirela Popa ◽  
Zsolt Ruzsics ◽  
Mark Lötzerich ◽  
Lars Dölken ◽  
Christopher Buser ◽  
...  
Keyword(s):  
Unit Length ◽  
Virus Production ◽  
Dominant Negative ◽  
Murine Cytomegalovirus ◽  
Phenotypic Characterization ◽  
Reading Frame ◽  
Viral Capsids ◽  
Gene Block ◽  
Nuclear Egress ◽  
C Terminus

ABSTRACT The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.

scholarly journals HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites

2016 ◽  
Vol 90 (15) ◽  
pp. 6709-6723 ◽  
Author(s):  
François M. Pujol ◽  
Vibor Laketa ◽  
Florian Schmidt ◽  
Markus Mukenhirn ◽  
Barbara Müller ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Virus Assembly ◽  
Adaptor Protein ◽  
Restriction Factor ◽  
Dependent Manner ◽  
Anterograde Transport ◽  
Particle Release ◽  
Hiv 1 ◽  
Tetherin Antagonism

ABSTRACTThe host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process.IMPORTANCECD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

scholarly journals Daxx Inhibits HIV-1 Reverse Transcription and Uncoating in a SUMO-Dependent Manner

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 636
Author(s):  
Sarah Maillet ◽  
Juliette Fernandez ◽  
Mathilde Decourcelle ◽  
Khadija El Koulali ◽  
Fabien P. Blanchet ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Transcriptional Repression ◽  
Protein S ◽  
Dependent Manner ◽  
Death Domain ◽  
Proteomic Screen ◽  
C Terminus ◽  
Biochemical Analyses ◽  
Hiv 1 ◽  
Dependent Interaction

Death domain-associated protein 6 (Daxx) is a multifunctional, ubiquitously expressed and highly conserved chaperone protein involved in numerous cellular processes, including apoptosis, transcriptional repression, and carcinogenesis. In 2015, we identified Daxx as an antiretroviral factor that interfered with HIV-1 replication by inhibiting the reverse transcription step. In the present study, we sought to unravel the molecular mechanism of Daxx-mediated restriction and, in particular, to identify the protein(s) that Daxx targets in order to achieve its antiviral activity. First, we show that the SUMO-interacting motif (SIM) located at the C-terminus of the protein is strictly required for Daxx to inhibit HIV-1 reverse transcription. By performing a quantitative proteomic screen combined with classical biochemical analyses, we found that Daxx associated with incoming HIV-1 cores through a SIM-dependent interaction with cyclophilin A (CypA) and capsid (CA). Daxx was found to reside within a multiprotein complex associated with viral capsids, also containing TNPO3, TRIM5α, and TRIM34. Given the well-known influence of these cellular factors on the stability of HIV-1 cores, we investigated the effect of Daxx on the cytoplasmic fate of incoming cores and found that Daxx prevented HIV-1 uncoating in a SIM-dependent manner. Altogether, our findings suggest that, by recruiting TNPO3, TRIM5α, and TRIM34 and possibly other proteins onto incoming HIV-1 cores through a SIM-dependent interaction with CA-bound CypA, Daxx increases their stability, thus preventing uncoating and reverse transcription. Our study uncovers a previously unknown function of Daxx in the early steps of HIV-1 infection and further illustrates how reverse transcription and uncoating are two tightly interdependent processes.

scholarly journals Cytoplasmic Cargo Receptor p62 Inhibits Avibirnavirus Replication by Mediating Autophagic Degradation of Viral Protein VP2

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Yahui Li ◽  
Boli Hu ◽  
Gang Ji ◽  
Yina Zhang ◽  
Chenyang Xu ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Viral Protein ◽  
Virus Assembly ◽  
Virus Production ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Virus Particles ◽  
Selective Autophagy ◽  
Cargo Receptor ◽  
Autophagic Degradation

ABSTRACT Selective autophagy regulates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and aggregated proteins. Furthermore, autophagy is capable of degrading avibirnavirus, but the mechanism responsible for this process is unclear. Here, we show that autophagy cargo receptor p62 regulates the degradation of the avibirnavirus capsid protein VP2. Binding of p62 to VP2 enhances autophagic induction and promotes autophagic degradation of viral protein VP2. Further study showed that the interaction of p62 with viral protein VP2 is dependent on ubiquitination at the K411 site of VP2 and the ubiquitin-associated domain of p62. Mutation analysis showed that the K411R mutation of viral protein VP2 prohibits its p62-mediated degradation. Consistent with this finding, p62 lacking the ubiquitin-associated domain or the LC3-interacting region no longer promoted the degradation of VP2. Virus production revealed that the knockout of p62 but not the overexpression of p62 promotes the replication of avibirnavirus. Collectively, our findings suggest that p62 mediates selective autophagic degradation of avibirnavirus protein VP2 in a ubiquitin-dependent manner and is an inhibitor of avibirnavirus replication. IMPORTANCE Avibirnavirus causes severe immunosuppression and mortality in young chickens. VP2, the capsid protein of avibirnavirus, is responsible for virus assembly, maturation, and replication. Previous study showed that avibirnavirus particles could be engulfed into the autophagosome and degradation of virus particles took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells.

scholarly journals Role of ESCRT-I in Retroviral Budding

2003 ◽  
Vol 77 (8) ◽  
pp. 4794-4804 ◽  
Author(s):  
Juan Martin-Serrano ◽  
Trinity Zang ◽  
Paul D. Bieniasz
Keyword(s):  
Leucine Zipper ◽  
Ebola Virus ◽  
Dominant Negative ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Dominant Negative Inhibitor ◽  
Retroviral Budding ◽  
Vacuolar Protein ◽  
Hiv 1 ◽  
Class E

ABSTRACT Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.

scholarly journals Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

2020 ◽  
Author(s):  
Seiya Ozono ◽  
Yanzhao Zhang ◽  
Minoru Tobiume ◽  
Satoshi Kishigami ◽  
Kenzo Tokunaga
Keyword(s):  
Virus Production ◽  
Luciferase Assay ◽  
Virus Particles ◽  
Protein Levels ◽  
C Terminus ◽  
P24 Protein ◽  
P24 Antigen ◽  
Routine Handling ◽  
Hiv 1 ◽  
Peptide Tag

ABSTRACTIn virological studies using HIV-1 proviral clones, virus production is normally monitored by either an RT assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. In addition, sample dilution is always required in ELISA to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. First, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the production or infectivity of the resultant viruses. Electron microscopy revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1 production.

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