scholarly journals An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

1999 ◽  
Vol 73 (5) ◽  
pp. 4127-4135 ◽  
Author(s):  
C. Helga-Maria ◽  
Marie-Louise Hammarskjöld ◽  
David Rekosh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Stem Structure

ABSTRACT Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5′ splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

scholarly journals Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Nuclear Localization Mediates Early Viral mRNA Expression

2002 ◽  
Vol 76 (20) ◽  
pp. 10444-10454 ◽  
Author(s):  
Jielin Zhang ◽  
Clyde S. Crumpacker
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mrna Expression ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleocapsid Protein ◽  
Viral Mrna ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 × 109 cells/day and 10.3 × 109 virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.

scholarly journals A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions

2000 ◽  
Vol 74 (24) ◽  
pp. 11811-11824 ◽  
Author(s):  
Kalpana Gupta ◽  
David Ott ◽  
Thomas J. Hope ◽  
Robert F. Siliciano ◽  
Jef D. Boeke
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Productive Infection ◽  
Genomic Rna ◽  
Virion Production ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55Gag and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4+ T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55Gag and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

scholarly journals Dissociation of Genome Dimerization from Packaging Functions and Virion Maturation of Human Immunodeficiency Virus Type 1

2002 ◽  
Vol 76 (3) ◽  
pp. 959-967 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Aikichi Iwamoto ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Rna ◽  
Rna Packaging ◽  
Virus Particles ◽  
Immunodeficiency Virus ◽  
Rna Interaction ◽  
Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is thought to play important roles at various stages of the virus life cycle. Recently we showed that the DIS/DLS region affects RNA-RNA interaction in intact virus particles, by demonstrating that duplication of the region in viral RNA caused the production of virus particles containing partially monomeric RNAs. We have extended this finding and succeeded for the first time in creating mutant particles which contain only monomeric RNAs without modifying any viral proteins. In terms of RNA encapsidation ability, virion density, and protein processing, the mutant particles were comparable to wild-type particles. The level of production of viral DNA by the mutant virus construct in infected cells was also comparable to that of the constructs that produced exclusively dimeric RNA, indicating that monomeric viral RNA could be the template for strand transfer. These results indicated that the RNA dimerization of HIV-1 could be separated from viral RNA packaging and was not absolutely required for RNA packaging, virion maturation, and reverse transcription.

scholarly journals Possible Role of Dimerization in Human Immunodeficiency Virus Type 1 Genome RNA Packaging

2003 ◽  
Vol 77 (7) ◽  
pp. 4060-4069 ◽  
Author(s):  
Jun-Ichi Sakuragi ◽  
Shigeharu Ueda ◽  
Aikichi Iwamoto ◽  
Tatsuo Shioda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Packaging ◽  
Dimer Formation ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Lower Stem ◽  
Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5′ region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.

scholarly journals Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

2003 ◽  
Vol 77 (10) ◽  
pp. 5547-5556 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Elena N. Chertova ◽  
Tracy D. Gagliardi ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Protease Activity ◽  
Genomic Rna ◽  
Wild Type ◽  
Virion Production ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four- to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10−6 relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

scholarly journals Human Immunodeficiency Virus Type 1 hnRNP A/B-Dependent Exonic Splicing Silencer ESSV Antagonizes Binding of U2AF65 to Viral Polypyrimidine Tracts

2003 ◽  
Vol 23 (23) ◽  
pp. 8762-8772 ◽  
Author(s):  
Jeffrey K. Domsic ◽  
Yibin Wang ◽  
Akila Mayeda ◽  
Adrian R. Krainer ◽  
C. Martin Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Independent Functions ◽  
Immunodeficiency Virus ◽  
U1 Snrnp ◽  
Precursor Rna ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3′ splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3′ splice sites. We further show evidence suggesting that U1 snRNP binds the 5′ splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5′ splice site interactions during virus replication are discussed.

scholarly journals Human Cellular Nucleic Acid-Binding Protein Zn2+ Fingers Support Replication of Human Immunodeficiency Virus Type 1 When They Are Substituted in the Nucleocapsid Protein

2003 ◽  
Vol 77 (15) ◽  
pp. 8524-8531 ◽  
Author(s):  
Connor F. McGrath ◽  
James S. Buckman ◽  
Tracy D. Gagliardi ◽  
William J. Bosche ◽  
Lori V. Coren ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Structural Characteristics ◽  
Nucleic Acid Binding ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn2+ fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn2+ fingers. The sequence of the NH2-terminal NC Zn2+ finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn2+ finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn2+ fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn2+ finger compared to mutant and wild-type Zn2+ fingers in NC that support replication. The present study shows that six of seven of the Zn2+ fingers from the CNBP protein can be used as substitutes for the Zn2+ finger in the NH2-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn2+ finger mutants.

scholarly journals The Double-Stranded RNA-Binding Protein Staufen Is Incorporated in Human Immunodeficiency Virus Type 1: Evidence for a Role in Genomic RNA Encapsidation

2000 ◽  
Vol 74 (12) ◽  
pp. 5441-5451 ◽  
Author(s):  
Andrew J. Mouland ◽  
Johanne Mercier ◽  
Ming Luo ◽  
Luc Bernier ◽  
Luc DesGroseillers ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Protein ◽  
Genomic Rna ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.

scholarly journals Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

2000 ◽  
Vol 74 (19) ◽  
pp. 8938-8945 ◽  
Author(s):  
Markus Dettenhofer ◽  
Shan Cen ◽  
Bradley A. Carlson ◽  
Lawrence Kleiman ◽  
Xiao-Fang Yu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA3 Lyswere additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced invif mutant virions. Moreover, purified vifmutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

Sign in / Sign up

Export Citation Format

Share Document