scholarly journals The Conformation of the Mature Dimeric Human Immunodeficiency Virus Type 1 RNA Genome Requires Packaging of Pol Protein

2002 ◽  
Vol 76 (9) ◽  
pp. 4331-4340 ◽  
Author(s):  
M. Shehu-Xhilaga ◽  
M. Hill ◽  
J. A. Marshall ◽  
J. Kappes ◽  
S. M. Crowe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Precursor Protein ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Rna Genome ◽  
Rna Dimer ◽  
Hiv 1

ABSTRACT The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

scholarly journals Proteolytic Processing of the P2/Nucleocapsid Cleavage Site Is Critical for Human Immunodeficiency Virus Type 1 RNA Dimer Maturation

2001 ◽  
Vol 75 (19) ◽  
pp. 9156-9164 ◽  
Author(s):  
M. Shehu-Xhilaga ◽  
H. G. Kraeusslich ◽  
S. Pettit ◽  
R. Swanstrom ◽  
J. Y. Lee ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cleavage Site ◽  
Proteolytic Processing ◽  
Immunodeficiency Virus ◽  
Rna Dimer ◽  
Primary Cleavage ◽  
Hiv 1

ABSTRACT Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

scholarly journals Infectious Molecular Clones with the Nonhomologous Dimer Initiation Sequences Found in Different Subtypes of Human Immunodeficiency Virus Type 1 Can Recombine and Initiate a Spreading Infection In Vitro

1998 ◽  
Vol 72 (5) ◽  
pp. 3991-3998 ◽  
Author(s):  
Daniel C. St. Louis ◽  
Deanna Gotte ◽  
Eric Sanders-Buell ◽  
David W. Ritchey ◽  
Mika O. Salminen ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Packaging ◽  
Immunodeficiency Virus ◽  
Rna Dimer ◽  
Initiation Sequence ◽  
Hiv 1

ABSTRACT Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.

scholarly journals DNA Repair Enzyme Uracil DNA Glycosylase Is Specifically Incorporated into Human Immunodeficiency Virus Type 1 Viral Particles through a Vpr-Independent Mechanism

1999 ◽  
Vol 73 (2) ◽  
pp. 1682-1688 ◽  
Author(s):  
Karen E. Willetts ◽  
Françoise Rey ◽  
Isabelle Agostini ◽  
Jean-Marc Navarro ◽  
Yves Baudat ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Repair ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Repair Enzyme ◽  
Uracil Dna Glycosylase ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.

scholarly journals Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding

2000 ◽  
Vol 74 (21) ◽  
pp. 10074-10080 ◽  
Author(s):  
Una O'Doherty ◽  
William J. Swiggard ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Target Cells ◽  
Virus Binding ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1IIIBvirions at 1,200 × g for 2 h at 25°C increases the number of particles that bind to CEM-SS T-cell targets by ∼40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37°C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, ∼100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24 gag . Because the modestg forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.

scholarly journals Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Its Own Translation

2006 ◽  
Vol 80 (21) ◽  
pp. 10478-10486 ◽  
Author(s):  
Emma C. Anderson ◽  
Andrew M. L. Lever
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Packaging Signal ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5′ untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5′ UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.

scholarly journals Identification of Staufen in the Human Immunodeficiency Virus Type 1 Gag Ribonucleoprotein Complex and a Role in Generating Infectious Viral Particles

2004 ◽  
Vol 24 (7) ◽  
pp. 2637-2648 ◽  
Author(s):  
Laurent Chatel-Chaix ◽  
Jean-Francois Clément ◽  
Catherine Martel ◽  
Véronique Bériault ◽  
Anne Gatignol ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Host Protein ◽  
Cell Fractionation ◽  
Genomic Rna ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55Gag, the principal mediator of HIV-1 genomic RNA encapsidation. Chimeric proviruses harboring wild-type or mutant forms of Staufen were expressed in 293T cells. Cell fractionation analyses demonstrated that Staufen cosedimented with pr55Gag within detergent-resistant, trypsin-sensitive complexes that excluded mature capsid and matrix proteins. Coimmunoprecipitation and bioluminescence resonance energy transfer assays demonstrated a specific and direct interaction between Staufen and the nucleocapsid domain of pr55Gag in vitro and in live cells. This interaction is shown here to be mediated by Staufen's dsRBD3, with a contribution from its C-terminal domain. Immunoprecipitation and reverse transcription-PCR analyses showed that the 9-kb genomic RNA was found within Staufen-containing immune complexes. Spliced HIV-1 RNAs were not detected in these Staufen complexes, indicating a preferential association of Staufen with the 9-kb species. These results substantiate that Staufen and pr55Gag interact directly during HIV-1 expression. Knockdown of Staufen expression by small interfering RNAs in HIV-1-expressing cells demonstrated that this cellular protein was important for the generation of infectious virus. These data show that Staufen, pr55Gag, and genomic RNA are part of the same intracellular complex and support a role for Staufen in pr55Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

scholarly journals Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

1997 ◽  
Vol 41 (5) ◽  
pp. 1017-1023 ◽  
Author(s):  
R W Humphrey ◽  
A Ohagen ◽  
D A Davis ◽  
T Fukazawa ◽  
H Hayashi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Polyprotein Processing ◽  
Gag Polyprotein ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Hiv 1

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

scholarly journals Human Immunodeficiency Virus Type 1-Like DNA Sequences and Immunoreactive Viral Particles with Unique Association with Breast Cancer

1998 ◽  
Vol 5 (5) ◽  
pp. 645-653 ◽  
Author(s):  
Eva M. Rakowicz-Szulczynska ◽  
Betty Jackson ◽  
Adriana M. Szulczynska ◽  
McClure Smith
Keyword(s):  
Breast Cancer ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dna Sequences ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by human immunodeficiency virus type 1 (HIV-1) and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. The binding of an epitope-specific anti-HIV-1 gp120 monoclonal antibody (MAb) (amino acids 308 to 322) to cancer RAK antigens has been found to be inhibited by a peptide derived from variable loop V3 of HIV-1. Breast cancer DNAs of 40 patients were PCR amplified with HIV-1 gp41-derived primers, and all of the samples were found to be positive. The DNA fragments amplified in seven blindly selected breast cancer samples were sequenced. The breast cancer DNA sequences showed at least 90% homology to the HIV-1 gene for gp41. Antisense oligonucleotides complementary to the HIV-1-like sequences inhibited reverse transcriptase activity and inhibited the growth of breast cancer cells in vitro. Viral particles detected in breast cancer cell lines were strongly immunogold labeled with the anti-HIV-1 gp120 MAb. The results obtained strongly suggest that the long-postulated breast cancer virus may, in fact, be related to HIV-1.

scholarly journals Gag-Pol Supplied in trans Is Efficiently Packaged and Supports Viral Function in Human Immunodeficiency Virus Type 1

2001 ◽  
Vol 75 (15) ◽  
pp. 6835-6840 ◽  
Author(s):  
M. K. Hill ◽  
C. W. Hooker ◽  
D. Harrich ◽  
S. M. Crowe ◽  
J. Mak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protein Translation ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
In Trans ◽  
Virion Formation ◽  
Hiv 1

ABSTRACT The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

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