A Conserved Mechanism within the 5'-Leader RNA Genome among HIV-1 and SIVcpz Strains during Viral Genome Recognition

The HIV-1 integrase enzyme (IN) plays a critical role in the viral life cycle by integrating the reverse-transcribed viral DNA into the host chromosome. This function of IN has been well studied, and the knowledge gained has informed the design of small molecule inhibitors that now form key components of antiretroviral therapy regimens. Recent discoveries unveiled that IN has an under-studied yet equally vital second function in human immunodeficiency virus type 1 (HIV-1) replication. This involves IN binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. Inhibition of IN binding to the viral RNA genome results in mislocalization of the viral genome inside the virus particle, and its premature exposure and degradation in target cells. The roles of IN in integration and virion morphogenesis share a number of common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers. Herein we describe these two functions of IN within the context of the HIV-1 life cycle, how IN binding to the viral genome is coordinated by the major structural protein, Gag, and discuss the value of targeting the second role of IN in virion morphogenesis.

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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 Role of the Stem-Loop A RNA Promoter in Flavivirus Replication

Viruses ◽

10.3390/v13061107 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1107

Author(s):

Kyung H. Choi

Keyword(s):

Viral Genome ◽

Rna Synthesis ◽

Current Model ◽

Viral Rna ◽

Genome Replication ◽

Stem Loop ◽

Genome Recognition ◽

Rna Genome ◽

Long Stem ◽

Rna Promoter

An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5′-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5′ guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5′ terminus of the viral genome to initiate RNA synthesis at the 3′ terminus.

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PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

Genome Biology ◽

10.1186/s13059-021-02307-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Maria Artesi ◽

Vincent Hahaut ◽

Basiel Cole ◽

Laurens Lambrechts ◽

Fereshteh Ashrafi ◽

...

Keyword(s):

Viral Genome ◽

Clonal Expansion ◽

Endogenous Retroviruses ◽

Viral Genomes ◽

Short Read Sequencing ◽

Long Reads ◽

Infected Cells ◽

Insertion Sites ◽

Viral Insertion ◽

Hiv 1

AbstractThe integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

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Circularization of the HIV-1 RNA genome

Nucleic Acids Research ◽

10.1093/nar/gkm564 ◽

2007 ◽

Vol 35 (15) ◽

pp. 5253-5261 ◽

Cited By ~ 27

Author(s):

M. Ooms ◽

T. E. M. Abbink ◽

C. Pham ◽

B. Berkhout

Keyword(s):

Rna Genome ◽

Hiv 1

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HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

10.1101/2021.01.16.426924 ◽

2021 ◽

Author(s):

Sanela Rankovic ◽

Akshay Deshpande ◽

Shimon Harel ◽

Christopher Aiken ◽

Itay Rousso

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Morphological Changes ◽

Viral Capsid ◽

Target Cells ◽

Viral Core ◽

Successful Infection ◽

Capsid Shell ◽

Hiv 1

AbstractThe HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

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Mutations in the Human Immunodeficiency Virus Type 1 Integrase D,D(35)E Motif Do Not Eliminate Provirus Formation

Journal of Virology ◽

10.1128/jvi.72.6.4678-4685.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4678-4685 ◽

Cited By ~ 34

Author(s):

Meenakshi Gaur ◽

Andrew D. Leavitt

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Genome ◽

Viral Dna ◽

Infectious Titer ◽

Immunodeficiency Virus ◽

Acidic Residues ◽

Hiv 1

ABSTRACT The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3′-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 103- to 104-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721–728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per μg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3′-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.

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HIV-1 regulatory genes and their role in the viral genome realization

Biopolymers and Cell ◽

10.7124/bc.0003b9 ◽

1994 ◽

Vol 10 (5) ◽

pp. 5-30

Author(s):

A. P. Kukharenko ◽

A. D. Shved

Keyword(s):

Viral Genome ◽

Regulatory Genes ◽

Hiv 1

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Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01075-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 491-501 ◽

Cited By ~ 51

Author(s):

Olivier Delelis ◽

Sylvain Thierry ◽

Frédéric Subra ◽

Françoise Simon ◽

Isabelle Malet ◽

...

Keyword(s):

Viral Genome ◽

Effective Concentration ◽

Coding Region ◽

Delayed Emergence ◽

Infected Cells ◽

High Level ◽

Emergence Of Resistance ◽

Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

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Electron Cryotomography Studies of Maturing HIV-1 Particles Reveal the Assembly Pathway of the Viral Core

Journal of Virology ◽

10.1128/jvi.02997-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1267-1277 ◽

Cited By ~ 42

Author(s):

Cora L. Woodward ◽

Sarah N. Cheng ◽

Grant J. Jensen

Keyword(s):

Viral Genome ◽

Virus Assembly ◽

Structural Characteristics ◽

Hexagonal Lattice ◽

The Body ◽

The Core ◽

Assembly Pathway ◽

Viral Particles ◽

Electron Cryotomography ◽

Hiv 1

ABSTRACTTo better characterize the assembly of the HIV-1 core, we have used electron cryotomography (ECT) to image infected cells and the viral particles cryopreserved next to them. We observed progressive stages of virus assembly and egress, including flower-like flat Gag lattice assemblies, hemispherical budding profiles, and virus buds linked to the plasma membrane via a thin membrane neck. The population of budded viral particles contains immature, maturation-intermediate, and mature core morphologies. Structural characteristics of the maturation intermediates suggest that the core assembly pathway involves the formation of a CA sheet that associates with the condensed ribonucleoprotein (RNP) complex. Our analysis also reveals a correlation between RNP localization within the viral particle and the formation of conical cores, suggesting that the RNP helps drive conical core assembly. Our findings support an assembly pathway for the HIV-1 core that begins with a small CA sheet that associates with the RNP to form the core base, followed by polymerization of the CA sheet along one side of the conical core toward the tip, and then closure around the body of the cone.IMPORTANCEDuring HIV-1 assembly and release, the Gag polyprotein is organized into a signature hexagonal lattice, termed the immature lattice. To become infectious, the newly budded virus must disassemble the immature lattice by proteolyzing Gag and then reassemble the key proteolytic product, the structural protein p24 (CA), into a distinct, mature hexagonal lattice during a process termed maturation. The mature HIV-1 virus contains a conical capsid that encloses the condensed viral genome at its wide base. Mutations or small molecules that interfere with viral maturation also disrupt viral infectivity. Little is known about the assembly pathway that results in the conical core and genome encapsidation. Here, we have used electron cryotomography to structurally characterize HIV-1 particles that are actively maturing. Based on the morphologies of core assembly intermediates, we propose that CA forms a sheet-like structure that associates with the condensed viral genome to produce the mature infectious conical core.

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