Cellular Proteo-Transcriptomic Changes in the Immediate Early-Phase of Lentiviral Transduction

Lentivirus-based vectors derived from human immunodeficiency viruses type 1 and 2 (HIV-1 and 2) are widely used tools in research and may also be utilized in clinical settings. Like their parental virions, they are known to depend on the cellular machinery for successful gene delivery and integration. While most of the studies on cellular proteomic and transcriptomic changes have focused on the late phase of the transduction, studies of those changes in early time-points, especially in the case of HIV-2 based vectors, are widely lacking. Using second generation HIV-1 and 2 vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vectors, we transduced HEK-293T human embryonic kidney cells and carried out transcriptomic profiling at 0 and 2 h time points, with accompanying proteomic analysis at 2 h following transduction. Significant variations were observed in gene expression profile between HIV-1 and HIV-2 transduced samples. Thrombospondin 1 (THBS1), collagens (COL1A2, COL3A1), and eukaryotic translation factors (EIF3CL) in addition to various genes coding for long non-coding RNA (lncRNA) were significantly upregulated 2 h after HIV-2 transduction compared to HIV-1. Label-free quantification mass spectrometry (MS) indicated that seven proteins involved in RNA binding, mRNA transport, and chaperoning were significantly downregulated. The identification of cellular protein targets of lentiviral vectors and their effect on the cellular transcriptome will undoubtedly shed more light on their complex life cycle and may be utilized against infection by their parental lentiviruses. Furthermore, characterizing the early phase of HIV-2 infection may aid in the understanding of its pathomechanism and long incubation period.

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Viral gene products and replication of the human immunodeficiency type 1 virus

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.5.c1135 ◽

1994 ◽

Vol 266 (5) ◽

pp. C1135-C1156 ◽

Cited By ~ 20

Author(s):

C. D. Morrow ◽

J. Park ◽

J. K. Wakefield

Keyword(s):

Host Cell ◽

Early Phase ◽

Viral Genome ◽

Social Issues ◽

Late Phase ◽

Viral Proteins ◽

Cell Surface Receptor ◽

Rna Genome ◽

Cell Chromosome ◽

Hiv 1

The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.

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The human immunodeficiency virus type 1 Rev protein: ménage à trois during the early phase of the lentiviral replication cycle

Journal of General Virology ◽

10.1099/vir.0.022509-0 ◽

2010 ◽

Vol 91 (8) ◽

pp. 1893-1897 ◽

Cited By ~ 11

Author(s):

Bastian Grewe ◽

Klaus Überla

Keyword(s):

Early Phase ◽

Late Phase ◽

Replication Cycle ◽

Main Function ◽

Human Immunodeficiency Viruses ◽

Cellular Cofactor ◽

Cell Genome ◽

Virus Replication Cycle ◽

Hiv 1 ◽

Rev Protein

The Rev protein of human immunodeficiency viruses (HIV) has long been recognized to be essential for the late phase of the virus replication cycle, due to its strong enhancement of expression of viral structural proteins. Surprisingly, a number of recent papers have demonstrated that Rev can also interfere with integration of the reverse-transcribed cDNA into the host-cell genome. This seems to be due to Rev's binding to integrase and LEDGF/p75, an important cellular cofactor of HIV-1 integration. As Rev is presumably expressed at sufficiently high levels only after the encoding genome has already integrated, the main function of Rev during the early phase might be to reduce genotoxicity due to excessive integration events after superinfection of the same cell by subsequent viruses. Other potential consequences for HIV-1 replication and evolution after co-infection of the same cell with two viruses are discussed.

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SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication

Journal of Virology ◽

10.1128/jvi.00693-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6275-6286 ◽

Cited By ~ 10

Author(s):

Sean Paz ◽

Michael L. Lu ◽

Hiroshi Takata ◽

Lydie Trautmann ◽

Massimo Caputi

Keyword(s):

Viral Replication ◽

Cell Viability ◽

Rna Binding ◽

Therapeutic Potential ◽

Cellular Protein ◽

Strong Inhibitor ◽

Minimal Impact ◽

Binding Domains ◽

Rna Binding Domains ◽

Hiv 1

ABSTRACTReplication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by >200-fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA-binding domains but not the arginine serine-rich activator domain can downregulate viral replication by >2,000-fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA-binding domains.IMPORTANCEMost drugs utilized to treat the HIV-1 infection are based on compounds that directly target proteins encoded by the virus. However, given the high viral mutation rate, the appearance of novel drug-resistant viral strains is common. Thus, there is a need for novel therapeutics with diverse mechanisms of action. In this study, we show that the cellular protein SRSF1 is a strong inhibitor of viral replication. Furthermore, expression of the SRSF1 RNA-binding domains alone can inhibit viral replication by >2,000-fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein, the RNA-binding domains, and the minimal effects observed on cell metabolism, further studies are warranted to assess the therapeutic potential of peptides derived from these sequences.

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Improved Stage Categorization of PTZ-Induced Kindling and Late Enhanced Neurogenesis in PTZ Kindled Mice

10.31661/gmj.v8i0.1511 ◽

2019 ◽

Vol 8 ◽

pp. 1511

Author(s):

Marzieh Shahpari ◽

Hadi Aligholi ◽

Mohammad Reza Namavar ◽

Farzaneh Vafaee ◽

Masoumeh Emamghoreishi

Keyword(s):

Subventricular Zone ◽

Early Phase ◽

Hind Limb ◽

Late Phase ◽

The Other ◽

Myoclonic Jerk ◽

Ptz Kindling ◽

First Occurrence ◽

Tonic Extension ◽

Behavioral Index

Background: There is no universally accepted behavioral scoring to define the early development of phenothiazine (PTZ) kindling. Therefore, studies investigating alterations of neurogenesis in the PTZ model were mainly focused on full kindled animals rather than early stages of kindling. This study aimed to determine an appropriate behavioral index for categorizing stages of PTZ kindling progress and to evaluate neurogenesis during PTZ kindling. Materials and Methods: Twenty-four mice were intraperitoneally injected with a sub convulsive dose of PTZ (40mg/kg) every other day until they became full kindled. The first occurrence of different seizure behaviors and their durations were recorded during kindling development, and the different stages of kindling were categorized. Neurogenesis was evaluated in the lateral subventricular zone (SVZ) at each stage of kindling by immunofluorescence staining. Results: First occurrence of restlessness, motionless staring, hind limb tonic extension, Straub’s tail, myoclonic jerk, and tonic-clonic were sequentially observed in more than 80% of animals with increasing PTZ injections. The duration of the myoclonic jerk was significantly longer than the other seizure behaviors. The significantly higher percentage of BrdU-positive cells was found in SVZ of mice showing tonic-clonic in comparison to other seizure behaviors. Conclusion: A hierarchy behavior was observed during the kindling process when considering the first occurrence of seizure behaviors. We defined the first occurrence of restlessness, motionless, hind limb tonic extension and Straub’s tail behaviors as an early phase, myoclonic jerk as a borderline phase and tonic-clonic as a late phase of PTZ-induced kindling. Our results indicated an enhanced SVZ neurogenesis at the late phase of kindling. [GMJ.2019;8:e1511]

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Nucleolin Improves Heart Function During Recovery From Myocardial Infarction by Modulating Macrophage Polarization

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248421989570 ◽

2021 ◽

pp. 107424842198957

Author(s):

Yuting Tang ◽

Xiaofang Lin ◽

Cheng Chen ◽

Zhongyi Tong ◽

Hui Sun ◽

...

Keyword(s):

Myocardial Infarction ◽

Early Phase ◽

Heart Function ◽

Macrophage Polarization ◽

Late Phase ◽

Macrophage Infiltration ◽

Enzyme Linked Immunosorbent Assay ◽

M2 Macrophage ◽

M2 Macrophage Polarization ◽

Nucleolin Expression

Background: Nucleolin has multiple functions within cell survival and proliferation pathways. Our previous studies have revealed that nucleolin can significantly reduce myocardial ischemia-reperfusion injury by promoting myocardial angiogenesis and reducing myocardial apoptosis. In this study, we attempted to determine the role of nucleolin in myocardial infarction (MI) injury recovery and the underlying mechanism. Methods: Male BALB/c mice aged 6–8 weeks were used to set up MI models by ligating the left anterior descending coronary artery. Nucleolin expression in the heart was downregulated by intramyocardial injection of a lentiviral vector expressing nucleolin-specific small interfering RNA. Macrophage infiltration and polarization were measured by real-time polymerase chain reaction, flow cytometry, and immunofluorescence. Cytokines were detected by enzyme-linked immunosorbent assay. Results: Nucleolin expression in myocardium after MI induction decreased a lot at early phase and elevated at late phase. Nucleolin knockdown impaired heart systolic and diastolic functions and decreased the survival rate after MI. Macrophage infiltration increased in the myocardium after MI. Most macrophages belonged to the M1 phenotype at early phase (2 days) and the M2 phenotype increased greatly at late phase after MI. Nucleolin knockdown in the myocardium led to a decrease in M2 macrophage polarization with no effect on macrophage infiltration after MI. Furthermore, Notch3 and STAT6, key regulators of M2 macrophage polarization, were upregulated by nucleolin in RAW 264.7 macrophages. Conclusions: Lack of nucleolin impaired heart function during recovery after MI by reducing M2 macrophage polarization. This finding probably points to a new therapeutic option for ischemic heart disease.

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Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein

Biosensors ◽

10.3390/bios11060180 ◽

2021 ◽

Vol 11 (6) ◽

pp. 180

Author(s):

Lucia Sarcina ◽

Giuseppe Felice Mangiatordi ◽

Fabrizio Torricelli ◽

Paolo Bollella ◽

Zahra Gounani ◽

...

Keyword(s):

Limit Of Detection ◽

Covalent Binding ◽

Hiv Infections ◽

Selectivity Ratio ◽

Selective Detection ◽

Label Free ◽

Self Assembled Monolayer ◽

Label Free Detection ◽

P24 Protein ◽

Hiv 1

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

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AFP and eGFR are related to early and late recurrence of HCC following antiviral therapy

BMC Cancer ◽

10.1186/s12885-021-08401-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Takao Watanabe ◽

Yoshio Tokumoto ◽

Kouji Joko ◽

Kojiro Michitaka ◽

Norio Horiike ◽

...

Keyword(s):

Risk Factors ◽

Confidence Interval ◽

Early Phase ◽

Late Phase ◽

Previous History ◽

Late Recurrence ◽

Early Recurrence ◽

Hazard Ratio ◽

Post Treatment ◽

Hcc Recurrence

Abstract Background An unexpected recurrence of hepatocellular carcinoma (HCC) sometimes occurs in patients with hepatitis C virus (HCV) after treatment with direct-acting antivirals (DAAs). However, the characteristics of patients with HCC recurrence may differ depending on time after DAA treatment. We aimed to identify risk factors related to HCC recurrence according to time after DAA treatment. Methods Of 1663 patients with HCV treated with a DAA, 199 patients had a previous history of HCC. We defined HCC recurrence within 1 year after DAA treatment as ‘early recurrence’, and recurrence more than 1 year after as ‘late recurrence’. The different risk factors between the early and late phases of HCC recurrence after the end of DAA therapy were investigated. Results Ninety-seven patients experienced HCC recurrence during the study period. Incidences of recurrence were 29.8, 41.0, and 53.4% at 1, 2, and 3 years, respectively, after the end of DAA therapy. Multivariate analysis identified post-treatment α-fetoprotein (AFP) as an independent factor contributing to HCC recurrence in the early phase (hazard ratio, 1.056; 95% confidence interval, 1.026–1.087, p < 0.001) and post-treatment estimated glomerular filtration rate (eGFR) (hazard ratio, 0.98; 95% confidence interval, 0.96–0.99, p = 0.032) as a predictor of HCC recurrence in the late phase. Conclusion Patients with higher post-treatment AFP in the early phase and those with lower post-treatment eGFR in the late phase had a high risk of HCC recurrence. The risk factors associated with HCC recurrence after DAA treatment were different between the early and late phases.

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High Natural Permissivity of Primary Rabbit Cells for HIV-1, with a Virion Infectivity Defect in Macrophages as the Final Replication Barrier

Journal of Virology ◽

10.1128/jvi.01607-10 ◽

2010 ◽

Vol 84 (23) ◽

pp. 12300-12314 ◽

Cited By ~ 14

Author(s):

Hanna-Mari Tervo ◽

Oliver T. Keppler

Keyword(s):

T Cells ◽

Late Phase ◽

Small Animal ◽

Receptor Complex ◽

Restriction Factors ◽

Cyclin T1 ◽

Primary Rabbit ◽

Species Specific ◽

Hiv 1

ABSTRACT An immunocompetent, permissive, small-animal model would be valuable for the study of human immunodeficiency virus type 1 (HIV-1) pathogenesis and for the testing of drug and vaccine candidates. However, the development of such a model has been hampered by the inability of primary rodent cells to efficiently support several steps of the HIV-1 replication cycle. Although transgenesis of the HIV receptor complex and human cyclin T1 have been beneficial, additional late-phase blocks prevent robust replication of HIV-1 in rodents and limit the range of in vivo applications. In this study, we explored the HIV-1 susceptibility of rabbit primary T cells and macrophages. Envelope-specific and coreceptor-dependent entry of HIV-1 was achieved by expressing human CD4 and CCR5. A block of HIV-1 DNA synthesis, likely mediated by TRIM5, was overcome by limited changes to the HIV-1 gag gene. Unlike with mice and rats, primary cells from rabbits supported the functions of the regulatory viral proteins Tat and Rev, Gag processing, and the release of HIV-1 particles at levels comparable to those in human cells. While HIV-1 produced by rabbit T cells was highly infectious, a macrophage-specific infectivity defect became manifest by a complex pattern of mutations in the viral genome, only part of which were deamination dependent. These results demonstrate a considerable natural HIV-1 permissivity of the rabbit species and suggest that receptor complex transgenesis combined with modifications in gag and possibly vif of HIV-1 to evade species-specific restriction factors might render lagomorphs fully permissive to infection by this pathogenic human lentivirus.

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