scholarly journals A versatile high throughput strategy for cloning the env gene of HIV-1

2020 ◽  
Author(s):  
Nitesh Mishra ◽  
Ayushman Dobhal ◽  
Shaifali Sharma ◽  
Kalpana Luthra
Keyword(s):  
High Throughput ◽  
Viral Evolution ◽  
Viral Envelope ◽  
Host Cell Membrane ◽  
Viral Neutralization ◽  
Gp120 Protein ◽  
Single Genome ◽  
Overlap Extension ◽  
Glycoprotein Gp120 ◽  
Hiv 1

AbstractThe trimeric envelope glycoprotein (gp120/gp41)3 of human immunodeficiency virus-1 (HIV-1) mediates viral and host cell membrane fusion, initiated by binding of viral envelope gp120 protein to the CD4 receptor on host immune cells. Functional env genes from infected individuals have been widely used as templates for vaccine design, for setting up viral neutralization assays and to study the viral evolution and pathogenesis. Traditional topoisomerase or T4 DNA polymerase mediated approaches for cloning single genome amplified (SGA) env genes are labor-intensive, cost-ineffective with low-throughput, thereby enabling functional analysis of only a limited number of env genes from the diverse circulating quasispecies in infected individuals. Herein, we report an efficient, easy to optimize and high-throughput approach for cloning diverse HIV-1 env genes. Multiple env/rev gene cassettes, derived from infected infants, were subjected to SGA using Phusion polymerase and utilized as megaprimers in overlap extension PCR mediated cloning (OEC), circumventing the requirement for novel enzymes. Furthermore, utilization of Phusion polymerase for both the amplification of env/rev cassettes and OEC allows convenient monitoring and optimization, thereby providing much greater flexibility and versatility for analysis of env genes from HIV-1 infected individuals.

scholarly journals Streamlined Subpopulation, Subtype, and Recombination Analysis of HIV-1 Half-Genome Sequences Generated by High-Throughput Sequencing

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Bhavna Hora ◽  
Naila Gulzar ◽  
Yue Chen ◽  
Konstantinos Karagiannis ◽  
Fangping Cai ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Vaccine Development ◽  
Consensus Sequence ◽  
Cost Effective ◽  
Recombination Analysis ◽  
Genome Sequences ◽  
Single Genome ◽  
Hiv 1

ABSTRACT High-throughput sequencing (HTS) has been widely used to characterize HIV-1 genome sequences. There are no algorithms currently that can directly determine genotype and quasispecies population using short HTS reads generated from long genome sequences without additional software. To establish a robust subpopulation, subtype, and recombination analysis workflow, we amplified the HIV-1 3′-half genome from plasma samples of 65 HIV-1-infected individuals and sequenced the entire amplicon (∼4,500 bp) by HTS. With direct analysis of raw reads using HIVE-hexahedron, we showed that 48% of samples harbored 2 to 13 subpopulations. We identified various subtypes (17 A1s, 4 Bs, 27 Cs, 6 CRF02_AGs, and 11 unique recombinant forms) and defined recombinant breakpoints of 10 recombinants. These results were validated with viral genome sequences generated by single genome sequencing (SGS) or the analysis of consensus sequence of the HTS reads. The HIVE-hexahedron workflow is more sensitive and accurate than just evaluating the consensus sequence and also more cost-effective than SGS. IMPORTANCE The highly recombinogenic nature of human immunodeficiency virus type 1 (HIV-1) leads to recombination and emergence of quasispecies. It is important to reliably identify subpopulations to understand the complexity of a viral population for drug resistance surveillance and vaccine development. High-throughput sequencing (HTS) provides improved resolution over Sanger sequencing for the analysis of heterogeneous viral subpopulations. However, current methods of analysis of HTS reads are unable to fully address accurate population reconstruction. Hence, there is a dire need for a more sensitive, accurate, user-friendly, and cost-effective method to analyze viral quasispecies. For this purpose, we have improved the HIVE-hexahedron algorithm that we previously developed with in silico short sequences to analyze raw HTS short reads. The significance of this study is that our standalone algorithm enables a streamlined analysis of quasispecies, subtype, and recombination patterns from long HIV-1 genome regions without the need of additional sequence analysis tools. Distinct viral populations and recombination patterns identified by HIVE-hexahedron are further validated by comparison with sequences obtained by single genome sequencing (SGS).

Human CD38 interferes with HIV‐1 fusion through a sequence homologous to the V3 loop of the viral envelope glycoprotein gp120.

2003 ◽  
Vol 17 (3) ◽  
pp. 1-20 ◽  
Author(s):  
Andrea Savarino ◽  
Thea Bensi ◽  
Annalisa Chiocchetti ◽  
Flavia Bottarel ◽  
Riccardo Mesturini ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
V3 Loop ◽  
Viral Envelope Glycoprotein ◽  
Envelope Glycoprotein Gp120 ◽  
Glycoprotein Gp120 ◽  
Hiv 1

scholarly journals Molecular dynamics for structural complexes of potential HIV-1 inhibitors with the viral envelope gp120 protein

2018 ◽  
Vol 62 (5) ◽  
pp. 576-584
Author(s):  
I. A. Kashyn ◽  
G. I. Nikolaev ◽  
M. A. Tuzikov ◽  
A. M. Andrianov
Keyword(s):  
Molecular Dynamics ◽  
Viral Envelope ◽  
Amino Acid Residues ◽  
Free Energies ◽  
Gp120 Protein ◽  
Dynamics Simulations ◽  
Hiv Drugs ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Envelope Gp120

Molecular dynamics simulations for the structural complexes of potential HIV-1 inhibitors with the viral envelope gp120 protein were carried out. Free energies of the formation of these supramolecular structures and contributions of individual amino-acid residues of gp120 to the enthalpy binding were calculated. The residues of gp120 critical for interactions with the ligands were identified. Based on the data obtained, five compounds promising for synthesis and testing for antiviral activity were selected. It is suggested that these compounds may be successfully used in the design of novel, potent and broad anti-HIV drugs.

scholarly journals Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1296
Author(s):  
Jonathan Burnie ◽  
Vera A. Tang ◽  
Joshua A. Welsh ◽  
Arvin T. Persaud ◽  
Laxshaginee Thaya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Single Cells ◽  
Surface Current ◽  
High Sensitivity ◽  
Viral Envelope ◽  
High Throughput Analysis ◽  
Associated Proteins ◽  
Hiv 1

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

scholarly journals HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Philip R. Tedbury ◽  
Mariia Novikova ◽  
Ayna Alfadhli ◽  
Yuta Hikichi ◽  
Ioannis Kagiampakis ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Leukemia Virus ◽  
Viral Evolution ◽  
Biological Significance ◽  
Viral Envelope ◽  
Trimer Formation ◽  
Compensatory Mutations ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation. IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.

Inhibition of HIV-1-Induced Cytopathogenicity, Syncytium Formation, and Virus-Cell Binding by Naphthalenedisulphonic Acids through Interaction with the Viral Envelope gp120 Glycoprotein

1993 ◽  
Vol 4 (4) ◽  
pp. 229-234 ◽  
Author(s):  
M. Baba ◽  
D. Schols ◽  
P. Mohan ◽  
E. De Clercq ◽  
S. Shigeta
Keyword(s):  
Monoclonal Antibody ◽  
Syncytium Formation ◽  
Viral Envelope ◽  
Flow Cytometric ◽  
Gp120 Antigen ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120 ◽  
Gp120 Glycoprotein ◽  
Hiv 1

Bis-naphthalenedisulphonic acid derivatives with a biphenyl spacer, 4,4′-[4,4′-biphenyldiylbis(sulphonyl-amino)]bis(5-hydroxy-2,7-naphthalenedisulphonic acid) and 3,3′-[4,4′-biphenyldiylbis(sulphonyl-amino)]bis(1,5-naphthalenedisulphonic acid), have previously been reported as potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in cell culture. These compounds have also proved inhibitory to syncytium formation in cocultures of MOLT-4 cells with HIV-1-infected HUT-78 cells. They also inhibit the binding of HIV-1 virions to MT-4 cells as determined by a flow cytometric (FACS) method. Further studies on their mechanism of action by the FACS have revealed that the compounds inhibit the binding of anti-gp120 monoclonal antibody to the viral envelope glycoprotein gp120. Binding of OKT4A/Leu3a monoclonal antibody to the cellular CD4 receptor is not affected by the compounds. These results suggest that the anti-HIV-1 activity of the naphthalenedisulphonic acid derivatives can be attributed to inhibition of the gp120-CD4 interaction through binding of the compounds to the viral gp120 antigen.

scholarly journals Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

2010 ◽  
Vol 84 (17) ◽  
pp. 8777-8789 ◽  
Author(s):  
Gregory Q. Del Prete ◽  
George J. Leslie ◽  
Beth Haggarty ◽  
Andrea P. O. Jordan ◽  
Josephine Romano ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Viral Envelope ◽  
Structural Basis ◽  
Molecular Pathways ◽  
V3 Loop ◽  
Cxcr4 Antagonist ◽  
Starting Point ◽  
Coreceptor Switch ◽  
Underlying Mechanisms ◽  
Hiv 1

ABSTRACT During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.

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