Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.

Incidence of Cytomegalovirus Infection After Repeat Keratoplasty and Associated Rate of Graft Failure.

INTRODUCTION: The aim of this work was to compare the prognosis and characteristics of patients with Cytomegalovirus (CMV) infection (CMV+) with those of patients without virus infection (Virus-) undergoing repeat keratoplasty. METHODS: This prospective propensity score-matched cohort study enrolled patients who underwent repeat keratoplasty for graft failure at the Peking University Third Hospital between January 2016 and May 2022. Patients with prior viral keratitis before the first keratoplasty were excluded. The primary outcome measure was the graft failure rate. The secondary outcome measures included the anterior segment characteristics, intraocular pressure (IOP), and endothelial cell density. RESULTS: Ninety-four matched patient pairs were included. The graft failure rate in the CMV+ group (71%) was higher than that in the Virus- group (29%) (P < 0.001). CMV infection in the cornea increased the risk of repeat graft failure and shortened the median survival time (hazard ratio, 3.876; 95% confidence intervals, 2.554-5.884; P < 0.001). The characteristics of graft failure included exacerbation of ocular surface inflammation, neovascularization, and opacification. Epithelial defects, high IOP, and endothelial decompensation were observed at an increased frequency in the CMV+ group (all P < 0.005). Recurrent CMV infection presented as early endothelial infection in the CMV+ group. Recurrence of CMV infection was confined to the graft endothelium without involving the stroma and epithelium post-repeat endothelial keratoplasty. CONCLUSIONS: CMV infection post-keratoplasty leads to persistent endothelial damage and graft opacification and significantly increases the risk of repeat graft failure. Localized recurrence of CMV infection in the endothelial grafts underscores the importance of monitoring and treatment. TRIAL REGISTRATION: Chictr.org.cn, ChiCTR1800014684.

PTEN Mediates the Silencing of Unintegrated HIV-1 DNA.

The integration of viral DNA into a host genome is an important step in HIV-1 replication. However, due to the high failure rate of integration, the majority of viral DNA exists in an unintegrated state during HIV-1 infection. In contrast to the robust expression from integrated viral DNA, unintegrated HIV-1 DNA is very poorly transcribed in infected cells, but the molecular machinery responsible for the silencing of unintegrated HIV-1 DNA remains poorly characterized. In this study, we sought to characterize new host factors for the inhibition of expression from unintegrated HIV-1 DNA. A genome-wide CRISPR-Cas9 knockout screening revealed the essential role of phosphatase and tensin homolog (PTEN) in the silencing of unintegrated HIV-1 DNA. PTEN's phosphatase activity negatively regulates the PI3K-Akt pathway to inhibit the transcription from unintegrated HIV-1 DNA. The knockout (KO) of PTEN or inhibition of PTEN's phosphatase activity by point mutagenesis activates Akt by phosphorylation and enhances the transcription from unintegrated HIV-1 DNA. Inhibition of the PI3K-Akt pathway by Akt inhibitor in PTEN-KO cells restores the silencing of unintegrated HIV-1 DNA. Transcriptional factors (NF-κB, Sp1, and AP-1) are important for the activation of unintegrated HIV-1 DNA in PTEN-KO cells. Finally, the knockout of PTEN increases the levels of active epigenetic marks (H3ac and H3K4me3) and the recruitment of PolII on unintegrated HIV-1 DNA chromatin. Our experiments reveal that PTEN targets transcription factors (NF-κB, Sp1, and AP-1) by negatively regulating the PI3K-Akt pathway to promote the silencing of unintegrated HIV-1 DNA.

In vitro characterization of cell-fusing agent virus DNA forms in Aedes aegypti mosquitoes.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

Vaccine development: Current trends and technologies.

Despite the effectiveness of vaccination in reducing or eradicating diseases caused by pathogens, there remain certain diseases and emerging infections for which developing effective vaccines is inherently challenging. Additionally, developing vaccines for individuals with compromised immune systems or underlying medical conditions presents significant difficulties. As well as traditional vaccine different methods such as inactivated or live attenuated vaccines, viral vector vaccines, and subunit vaccines, emerging non-viral vaccine technologies, including viral-like particle and nanoparticle vaccines, DNA/RNA vaccines, and rational vaccine design, offer new strategies to address the existing challenges in vaccine development. These advancements have also greatly enhanced our understanding of vaccine immunology, which will guide future vaccine development for a broad range of diseases, including rapidly emerging infectious diseases like COVID-19 and diseases that have historically proven resistant to vaccination. This review provides a comprehensive assessment of emerging non-viral vaccine production methods and their application in addressing the fundamental and current challenges in vaccine development.

Structural insights into the assembly and mechanism of mpox virus DNA polymerase complex F8-A22-E4-H5.

The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.

Detection of varicella zoster virus DNA in blood from immunocompromised patients during the week preceding the onset of herpes zoster rash.

OBJECTIVE: To assess whether varicella zoster virus (VZV) DNA can be detected in blood before herpes zoster (HZ) rash onset. METHOD: Monocentric retrospective study from January 2019 to March 2023 including patients with HZ and stored blood samples performed during the week preceding the onset of HZ rash. Blood samples were retrospectively analyzed for VZV DNA by quantitative PCR. RESULTS: Among the 138 patients with HZ during the study period, stored blood samples performed during the week preceding the onset of HZ rash were available for 13 of them. Twelve (92 %) patients were immunosuppressed, mostly due to solid organ transplantation (38 %), solid malignancy (31 %) or autoimmune disease (23 %). During the week preceding HZ onset, VZV DNA was detected in blood from 10 (77 %) patients, with a median value of 3.6 log (copies/mL) (IQR 3.3-3.9). At the time of HZ onset, all VZV PCR performed in available blood samples were positive. CONCLUSION: Our findings demonstrates that VZV DNA can be commonly detected in blood from immunocompromised patients during the prodromal phase of HZ. Early screening of VZV DNA in blood from high-risk immunocompromised patients might improve HZ therapeutic management.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells.

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

Prevalence of mpox viral DNA in cutaneous specimens of monkeypox-infected patients: a systematic review and meta-analysis.

Background: Human monkeypox (mpox) disease is a multicountry outbreak driven by human-human transmission which has resulted in an international public health emergency. However, there is limited evidence on the positivity rate of skin lesions for mpox viral DNA. We aim to fill this gap by estimating the pooled positivity rate of skin samples with mpox viral DNA from mpox patients globally. Methods: In this systematic review and meta-analysis, seven databases and several preprint servers have been extensively searched until 17 January 2023 according to a prospectively registered protocol (PROSPERO: CRD42023392505). Articles including the positivity rate of skin samples with mpox viral DNA in mpox-confirmed patients were considered eligible. After a quality assessment, a random-effect meta-analysis was used for pooled prevalence. To explore and resolve heterogeneity, we used statistical methods for outlier detection, influence analysis, and sensitivity analysis. Findings: Among the 331 articles retrieved after deduplication, 14 studies were finally included. The pooled positivity rate of the skin samples was 98.77% (95% CI: 94.74%-99.72%). After the removal of an influential outlier, I 2 for heterogeneity dropped from 92.5% to 10.8%. Meta-regression did not reveal any significant moderator. Conclusion/interpretation: The present findings reinforce that skin lesions act as a reservoir of mpox viral DNA and contribute to a high infectivity risk. This may be a prevailing basis of prompt transmission during the current multicountry outbreak and also needs further investigation. The present imperative outcome may benefit in producing valuable preventive and management procedures in an appropriate health strategy.

Revolving ATPase motors as asymmetrical hexamers in translocating lengthy dsDNA via conformational changes and electrostatic interactions in phi29, T7, herpesvirus, mimivirus, E. coli, and Streptomyces.

Investigations of the parallel architectures of biomotors in both prokaryotic and eukaryotic systems suggest a similar revolving mechanism in the use of ATP to drive translocation of the lengthy double-stranded (ds)DNA genomes. This mechanism is exemplified by the dsDNA packaging motor of bacteriophage phi29 that operates through revolving but not rotating dsDNA to "Push through a one-way valve". This unique and novel revolving mechanism discovered in phi29 DNA packaging motor was recently reported in other systems including the dsDNA packaging motor of herpesvirus, the dsDNA ejecting motor of bacteriophage T7, the plasmid conjugation machine TraB in Streptomyces, the dsDNA translocase FtsK of gram-negative bacteria, and the genome-packaging motor in mimivirus. These motors exhibit an asymmetrical hexameric structure for transporting the genome via an inch-worm sequential action. This review intends to delineate the revolving mechanism from a perspective of conformational changes and electrostatic interactions. In phi29, the positively charged residues Arg-Lys-Arg in the N-terminus of the connector bind the negatively charged interlocking domain of pRNA. ATP binding to an ATPase subunit induces the closed conformation of the ATPase. The ATPase associates with an adjacent subunit to form a dimer facilitated by the positively charged arginine finger. The ATP-binding induces a positive charging on its DNA binding surface via an allostery mechanism and thus the higher affinity for the negatively charged dsDNA. ATP hydrolysis induces an expanded conformation of the ATPase with a lower affinity for dsDNA due to the change of the surface charge, but the (ADP+Pi)-bound subunit in the dimer undergoes a conformational change that repels dsDNA. The positively charged lysine rings of the connector attract dsDNA stepwise and periodically to keep its revolving motion along the channel wall, thus maintaining the one-way translocation of dsDNA without reversal and sliding out. The finding of the presence of the asymmetrical hexameric architectures of many ATPases that use the revolving mechanism may provide insights into the understanding of translocation of the gigantic genomes including chromosomes in complicated systems without coiling and tangling to speed up dsDNA translocation and save energy.

Unravelling the mechanisms by which chronic hepatitis B infection is associated with an increased risk of gestational diabetes.

An independent association between chronic hepatitis B virus (HBV) and the development of gestational diabetes (GDM) has been reported in the literature. Ethnic background and regional influences have been demonstrated to play a role in the reporting of incidence rates of GDM among women with chronic HBV. The mechanisms behind this association are poorly understood, but evidence suggests an inflammatory basis. Viral factors such as chronic HBV replication, quantifiable by HBV viral load, have been proposed to contribute to the increasing risk of insulin resistance in pregnancy. More research is needed to better characterise the association and determine if any interventions early in pregnancy for women infected with chronic HBV would mitigate the development of GDM.

Serological and Molecular Survey on Domestic Dog Hepadnavirus in Household Dogs, Italy.

The discovery of hepadnaviruses in cats (domestic cat hepadnavirus, DCH) and of a DCH-like virus in dogs has raised several questions regarding the role of these viruses in pets, with particular emphasis on their potential impact on animal health and epidemiology, as well as possible zoonotic implications. In this study, by screening an age-stratified collection of 600 canine serum samples for DCH with an ELISA assay based on the recombinant core antigen (DCHCAg), specific antibodies were found with an overall prevalence of 10.0% (60/600), with a higher prevalence in younger and older dogs. By retesting the canine DCHCAbs-positive sera with an ELISA test based on the recombinant surface protein of DCH (DCHSAg), a total of 18 sera (30%, 18/60) also contained IgG anti-DCHSAg. All the sera were also assessed molecularly using either a consensus hepadnavirus PCR or a specific real-time PCR for DCH. Hepadnavirus DNA was detected in four seronegative dogs, with a prevalence rate of 0.7% (4/600). On sequence analysis of the polymerase region amplified with pan-hepadnavirus primers, the amplicons displayed the highest nucleotide identity (97.3-99.6%) to DCH sequences detected in cats and to the domestic dog hepadnavirus recently identified in a canine serum sample from Italy.

Nosocomial transmission of MPOX virus to health care workers -an emerging occupational hazard: A case report and review of the literature.

We present an unusual case of monkeypox (MPOX) virus transmission to a dermatology resident during examination of affected patients. Viral DNA sequencing led to the identification of the most likely contact. This case, along with a review of all published cases so far, emphasizes the possible hazard of MPOX transmission to health care personnel, even when wearing personal protective equipment. It also emphasizes the need for maintaining high index of suspicion when examining patients with new dermatological lesions and strict compliance with the revised Centers for Disease Control and Prevention recommendations for specimen collection from such patients.

Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion.

BACKGROUND: This study investigated the effect of nucleos(t)ide analogue (NUC) treatment on hepatitis B virus (HBV) DNA integration and hepatocyte clonal expansion, both of which are implicated in hepatocellular carcinoma (HCC) in chronic hepatitis B. METHODS: Twenty-eight patients receiving NUCs (11 lamivudine, 7 telbivudine, 10 entecavir) were included. All had liver biopsies at baseline and year 1, and 7 had a third biopsy at year 10. HBV DNA integration and hepatocyte clone size were assessed by inverse polymerase chain reaction. RESULTS: All patients had detectable HBV integration at baseline, with a median integration frequency of 1.01 × 109 per liver and hepatocyte clone size of 2.41 × 105. Neither integration frequency nor hepatocyte clone size correlated with age and HBV virologic parameters. After 1 year of treatment, HBV integration was still detectable in all patients, with a median of 5.74 × 108 integration per liver (0.22 log reduction; P = .008) and hepatocyte clone size of 1.22 × 105 (0.40 log reduction; P = .002). HBV integration remained detectable at year 10 of treatment, with a median integration frequency of 4.84 × 107 integration per liver (0.93 log reduction from baseline) and hepatocyte clone size of 2.55 × 104 (1.02 log reduction from baseline). From baseline through year 1 to year 10, there was a decreasing trend in both integration frequency and hepatocyte clone size (P = .066 and.018, respectively). CONCLUSIONS: NUCs reduced both HBV DNA integration and hepatocyte clonal expansion, suggesting another alternative pathway besides direct viral suppression to reduce HCC risk. Our findings supported the notion for a long-term NUC treatment to prevent HCC.

Molecular and Biochemical Assessment of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy.

Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease. Adeno-associated virus (AAV) mediated gene replacement, and CRISPR/Cas9-mediated genome editing hold the potential to treat DMD. Molecular and biochemical analyses are essential to determine gene transfer efficiency and therapeutic efficacy. In this chapter, we present a series of methods routinely used in our laboratory to extract and quantify DNA, RNA, and protein in gene therapy studies performed in the canine DMD model.

Multiple traces of monkeypox detected in non-sewered wastewater with sparse sampling from a densely populated metropolitan area in Asia.

The monkeypox virus is excreted in the feces of infected individuals. Therefore, there is an interest in using viral load detection in wastewater for sentinel early surveillance at a community level and as a complementary approach to syndromic surveillance. We collected wastewater from 63 sewered and non-sewered locations in Bangkok city center between May and August 2022. Monkeypox viral DNA copy numbers were quantified using real-time polymerase chain reaction (PCR) and confirmed positive by Sanger sequencing. Monkeypox viral DNA was first detected in wastewater from the second week of June 2022, with a mean copy number of 16.4 copies/ml (n = 3). From the first week of July, the number of viral DNA copies increased to a mean copy number of 45.92 copies/ml. Positive samples were Sanger sequenced and confirmed the presence of the monkeypox virus. Our study is the first to detect monkeypox viral DNA in wastewater from various locations within Thailand. Results suggest that this could be a complementary source for detecting viral DNA and predicting upcoming outbreaks.

Two-Color CRISPR Imaging Reveals Dynamics of Herpes Simplex Virus 1 Replication Compartments and Virus-Host Interactions.

Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.

Recent Development in Plasmonic Nanobiosensors for Viral DNA/RNA Biomarkers.

Recently, due to the coronavirus pandemic, the need for early diagnosis of infectious diseases, including viruses, is emerging. Though early diagnosis is essential to prevent infection and progression to severe illness, there are few technologies that accurately measure low concentrations of biomarkers. Plasmonic nanomaterials are attracting materials that can effectively amplify various signals, including fluorescence, Raman, and other optical and electromagnetic output. In this review, we introduce recently developed plasmonic nanobiosensors for measuring viral DNA/RNA as potential biomarkers of viral diseases. In addition, we discuss the future perspective of plasmonic nanobiosensors for DNA/RNA detection. This review is expected to help the early diagnosis and pathological interpretation of viruses and other diseases.

Different Pathways Conferring Integrase Strand-Transfer Inhibitors Resistance.

Integrase Strand Transfer Inhibitors (INSTIs) are currently used as the most effective therapy in the treatment of human immunodeficiency virus (HIV) infections. Raltegravir (RAL) and Elvitegravir (EVG), the first generation of INSTIs used successfully in clinical treatment, are susceptible to the emergence of viral resistance and have a high rate of cross-resistance. To counteract these resistant mutants, second-generation INSTI drugs have been developed: Dolutegravir (DTG), Cabotegravir (CAB), and Bictegravir (BIC). However, HIV is also able to develop resistance mechanisms against the second-generation of INSTIs. This review describes the mode of action of INSTIs and then summarizes and evaluates some typical resistance mutations, such as substitution and insertion mutations. The role of unintegrated viral DNA is also discussed as a new pathway involved in conferring resistance to INSTIs. This allows us to have a more detailed understanding of HIV resistance to these inhibitors, which may contribute to the development of new INSTIs in the future.