Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

Davis, Adam; Carr, Jillian M.; Bagley, Christopher J.; Powell, Jason A.; Warrilow, David; Harrich, David; Burrell, Christopher J.; Li, Peng

doi:10.1186/1742-4690-5-115

Cited by 10 publications

(12 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Do the sign of the net charge of the target protein also dominate the detection of our sensors? The isoelectric points ( pI s) of HIV-1 RT, CEA, NT-proBNP, and CRP are reported as 8~9 30 , 4.7 31 , 8.5 32 , and 7.4 33 , respectively. From our results, HIV-1 RT, CEA, and NT-proBNP cause current gain to drop from baseline as their concentration increase, while CRP causes current gain increase (Figs 2, 3 and 4).…”

Section: Resultsmentioning

confidence: 99%

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum

Chu

Sarangadharan

Regmi

et al. 2017

Sci Rep

192

145

View full text Add to dashboard Cite

In this study, a new type of field-effect transistor (FET)-based biosensor is demonstrated to be able to overcome the problem of severe charge-screening effect caused by high ionic strength in solution and detect proteins in physiological environment. Antibody or aptamer-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) are used to directly detect proteins, including HIV-1 RT, CEA, NT-proBNP and CRP, in 1X PBS (with 1%BSA) or human sera. The samples do not need any dilution or washing process to reduce the ionic strength. The sensor shows high sensitivity and the detection takes only 5 minutes. The designs of the sensor, the methodology of the measurement, and the working mechanism of the sensor are discussed and investigated. A theoretical model is proposed based on the finding of the experiments. This sensor is promising for point-of-care, home healthcare, and mobile diagnostic device.Field-effect transistors (FETs) attract great interest for biomolecular detection, due to their high sensitivity, small size, and label-free detection, which are suitable for point-of-care or personal homecare devices. Either planar or nanowire FET-based biosensors have been widely studied using various materials, such as Si 1 , GaN 2 , carbon nanotube (CNT) 3 , or graphene oxide 4 . Conventionally, FET-based biosensors with receptors (ex. antibody) immobilized on the gate region above the active channel of the FETs face an intrinsic issue, which is the severe charge screening effect in high ionic strength solutions, such as in serum or blood samples, leading to low sensitivity for direct detection of protein in the physiological environment. The Debye length in physiological salt environment (1X PBS) is near 0.7 nm, which is much smaller than the size of a regular IgG antibody (5~10 nm) 5 . In order to effectively detect proteins with receptor-immobilized FETs, the electrical measurements are usually conducted in diluted buffer solutions, such as in 0.1X PBS or 0.01X PBS, where the Debye lengths as 2.4 nm and 7.4 nm, respectively 1,6,7 . However, diluted ionic strength solution may cause the change in protein structure, resulting in the loss of protein activity, and the binding affinity as well. For most biological reactions, which occur in physiological high salt environment, a biosensor that can be used directly with physiological samples is much favored. Besides, an additional washing process is needed for conventional FET-based biosensors to remove the unbound antigen before electrical measurement, which also increases the complexity of the whole sensor system. Therefore, direct detection of the target protein in physiological sample is very demanding.Previously, several groups have reported that conventional FET-based biosensors can effectively detect proteins in physiological salt environment, using alternative current (AC) signals in drain-source voltage (V ds ), in conjunction with a reference electrode, in a relatively high frequency [8][9][10][11] . The better sensitivity of AC signals compared to that o...

show abstract

Section: Resultsmentioning

confidence: 99%

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum

Chu

Sarangadharan

Regmi

et al. 2017

Sci Rep

192

145

View full text Add to dashboard Cite

show abstract

“…Similar to HIV polyprotein processing [ 38 , 39 ], multiple protease cleavage steps produce intermediate protein products, before each RT isoform is released from the polyprotein. Active RT enzymes are generally heterodimers comprised of a large catalytic RT isoform containing an RNase H domain and a smaller RT isoform without the RNase H domain, which plays a structural role [ 40 , 45 , 46 ]. Figure 1 C shows the formation of two different sized ERVK RT isoforms.…”

Section: Resultsmentioning

confidence: 99%

“…Image Lab software [ 36 ] was used to determine the molecular weight of each band, as well as their density relative to that of the negative control. The identity of each band was predicted based on the molecular weight of each ERVK protein [ 37 ] and informed by the gag-pro-pol processing pattern of HIV [ 38 , 39 ], including HIV protein post-translational modifications [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

ERVK Polyprotein Processing and Reverse Transcriptase Expression in Human Cell Line Models of Neurological Disease

Manghera

Ferguson

Douville

2015

Viruses

View full text Add to dashboard Cite

Enhanced expression of the reverse transcriptase (RT) protein encoded by human endogenous retrovirus-K (ERVK) is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX) and astrocytic (SVGA) models of neuroinflammatory disease. Since the pro-inflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure—and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease.

show abstract

“…The p66 fingers subdomain made additional contacts to the vRNA:tR-NA(Lys3) complex and while the precise RNA contact is unknown, a likely target would include regions in U5 just ahead of the tRNA primer. Recently, different RT isoforms were detected in virions and in cells after infection suggesting that post-translational modifications could be important for activation [43]. Nucleotides are also implicated as a factor simply because intact virions can be induced to reverse transcribe in vitro by their addition.…”

Section: Entry To the Cytoplasm: The Pre-initiation Complex And Activmentioning

confidence: 98%

Maturation of the HIV reverse transcription complex: putting the jigsaw together

Warrilow

Tachedjian

Harrich

2009

Reviews in Medical Virology

Self Cite

View full text Add to dashboard Cite

Upon HIV attachment, fusion and entry into the host cell cytoplasm, the viral core undergoes rearrangement to become the mature reverse transcription complex (RTC). Reduced infectivity of viral deletion mutants of the core proteins, capsid and negative factor (Nef), can be complemented by vesicular stomatitis virus (VSV) pseudotyping suggesting a role for these viral proteins in a common event immediately post-entry. This event may be necessary for correct trafficking of the early complex. Enzymatic activation of the complex occurs either before or during RTC maturation, and may be dependent on the presence of deoxynucleotides in the host cell. The RTC initially becomes enlarged immediately after entry, which is followed by a decrease in its sedimentation rate consistent with core uncoating. Several HIV proteins associated with the RTC and recently identified host-cell proteins are important for reverse transcription while genome-wide siRNA knockdown studies have identified additional host cell factors that may be required for reverse transcription. Determining precisely how these proteins assist the RTC function needs to be addressed.

show abstract

Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

Cited by 10 publications

References 41 publications

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum

ERVK Polyprotein Processing and Reverse Transcriptase Expression in Human Cell Line Models of Neurological Disease

Maturation of the HIV reverse transcription complex: putting the jigsaw together

Contact Info

Product

Resources

About