scholarly journals Human Immunodeficiency Virus Type 1 Vif Protein Is Packaged into the Nucleoprotein Complex through an Interaction with Viral Genomic RNA

2001 ◽  
Vol 75 (16) ◽  
pp. 7252-7265 ◽  
Author(s):  
Mohammad A. Khan ◽  
Claudia Aberham ◽  
Sandra Kao ◽  
Hirofumi Akari ◽  
Robert Gorelick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Nucleoprotein Complex ◽  
Vif Protein ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

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.

scholarly journals Gag–Pol bearing a reverse transcriptase drug-resistant mutation influences viral genomic RNA incorporation into human immunodeficiency virus type 1 particles

2006 ◽  
Vol 87 (9) ◽  
pp. 2669-2677
Author(s):  
Renato S. Aguiar ◽  
Helena S. Pereira ◽  
Luciana J. Costa ◽  
Rodrigo M. Brindeiro ◽  
Amilcar Tanuri
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Wild Type Counterpart ◽  
Drug Resistant Mutation ◽  
Viral Genomic Rna ◽  
Hiv 1

The unspliced human immunodeficiency virus type 1 (HIV-1) RNA is both the messenger for Gag and Gag–Pol and the viral genomic RNA (vRNA) that is packaged into the virion. Although Gag alone is sufficient for the incorporation of vRNA into virus particles, Gag–Pol molecules play an important role in vRNA dimerization and virion maturation. Here, a cis model for vRNA packaging was demonstrated, in which nascent Gag–Pol molecules were preferentially co-encapsulated with their cognate RNA used as the template. Genome-incorporation frequencies were evaluated for two distinct HIV-1 proviral clones differing in their ability to respond to nevirapine (NVP) treatment in one round of infection. It was shown that, under NVP selection, there was a twofold-higher incorporation of vRNAs and integration of provirus genome carrying NVP resistance when compared with the wild-type counterpart. Although cis incorporation has been already demonstrated for Gag, the novelty of these findings is that newly acquired resistant mutations in Gag–Pol will select their specific genomic RNA during virus replication, thus rapidly increasing the chance of the emergence of resistant viruses during the course of anti-retroviral treatment.

scholarly journals Amino-Terminal Region of the Human Immunodeficiency Virus Type 1 Nucleocapsid Is Required for Human APOBEC3G Packaging

2004 ◽  
Vol 78 (21) ◽  
pp. 11841-11852 ◽  
Author(s):  
Kun Luo ◽  
Bindong Liu ◽  
Zuoxiang Xiao ◽  
Yunkai Yu ◽  
Xianghui Yu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Terminal Region ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Viral Genomic Rna ◽  
Hiv 1 ◽  
Human Apobec3g

ABSTRACT APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.

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 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 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.

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