scholarly journals HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 54 ◽  
Author(s):  
Fu-Hsien Yu ◽  
Kuo-Jung Huang ◽  
Chin-Tien Wang
Keyword(s):  
Gene Expression ◽  
Molecular Basis ◽  
Virus Assembly ◽  
Foamy Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Ribosomal Frameshifting ◽  
2A Peptide ◽  
Hiv 1 ◽  
Viral Enzyme

The pol retrovirus gene encodes required enzymes for virus replication and maturation. Unlike HIV-1 Pol (expressed as a Gag–Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag–Pol polyproteins. We placed a “self-cleaving” 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag–Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag–Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.

scholarly journals Visualizing the translation and packaging of HIV-1 full-length RNA

2020 ◽  
Vol 117 (11) ◽  
pp. 6145-6155 ◽  
Author(s):  
Jianbo Chen ◽  
Yang Liu ◽  
Bin Wu ◽  
Olga A. Nikolaitchik ◽  
Preeti R. Mohan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Rna Binding ◽  
Virus Assembly ◽  
Full Length ◽  
Rna Molecules ◽  
Rna Translation ◽  
Translation Template ◽  
A Genome ◽  
Hiv 1

HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation.

scholarly journals Human Rhinovirus Type 16: Mutant V1210A Requires Capsid-Binding Drug for Assembly of Pentamers To Form Virions during Morphogenesis

2003 ◽  
Vol 77 (11) ◽  
pp. 6235-6244 ◽  
Author(s):  
Wai-Ming Lee ◽  
Wensheng Wang
Keyword(s):  
Cdna Clone ◽  
Virus Assembly ◽  
Culture Collection ◽  
Human Rhinovirus ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Virus Infectivity ◽  
Wild Type ◽  
Drug Dependent

ABSTRACT Our laboratory has previously reported isolation of human rhinovirus type 16 (HRV16) mutants which depend on WIN 52035 to grow. A rapid rise of progeny virus infectivity occurred when drug was added late in growth cycles, suggesting that the drug-dependence lesion was at the step of virus assembly (W. Wang et al., J. Virol. 72:1210-1218, 1998). Here, we report that capsid subunits, 5S protomers and 14S pentamers, of a drug-dependent mutant were produced normally in the absence of drug, but mutant 80S empty capsids and 150S provirions were not formed, maturation cleavage of provirions (VP0 → VP2 + VP4) did not occur, and the unassembled mutant capsid subunits were degraded with a half-life of 15 min. Drug was not required by mutant virus for attachment, uncoating, RNA synthesis and protein synthesis, and polyprotein processing except maturation cleavage. The requirement of drug for assembly of mutant pentamers to form provirions and the rapid assembly of preformed subunits (synthesized in the absence of drug) after drug addition suggested that after native pentamers (P5) have been formed they must be converted to an assembly active state (P5∗), possibly by a conformational change induced by the binding of drug. We propose that pocket factor plays the same role in wild-type virus. In addition, we also report the construction and the properties of a full-length cDNA clone of HRV16, pR16.11, which produces in vitro transcripts with infectivity similar to that of virion RNA. This cDNA clone is available at the American Type Culture Collection.

scholarly journals The Human Immunodeficiency Virus Type 1 Vif Protein Reduces Intracellular Expression and Inhibits Packaging of APOBEC3G (CEM15), a Cellular Inhibitor of Virus Infectivity

2003 ◽  
Vol 77 (21) ◽  
pp. 11398-11407 ◽  
Author(s):  
Sandra Kao ◽  
Mohammad A. Khan ◽  
Eri Miyagi ◽  
Ron Plishka ◽  
Alicia Buckler-White ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Infectivity ◽  
Wild Type ◽  
Virus Particles ◽  
Immunodeficiency Virus ◽  
Intracellular Expression ◽  
Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

scholarly journals The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines

2018 ◽  
Vol 92 (21) ◽  
Author(s):  
Keisuke Nakagawa ◽  
Krishna Narayanan ◽  
Masami Wada ◽  
Vsevolod L. Popov ◽  
Maria Cajimat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Virus Assembly ◽  
Host Gene ◽  
Wild Type Virus ◽  
Rna Cleavage ◽  
Host Gene Expression ◽  
Biological Functions ◽  
Wild Type ◽  
Infected Cells

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induceIFN-β andIFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.IMPORTANCEMERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

scholarly journals Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity

1998 ◽  
Vol 72 (6) ◽  
pp. 4765-4774 ◽  
Author(s):  
Steve S.-L. Chen ◽  
Sheau-Fen Lee ◽  
Huey-Jong Hao ◽  
Chin-Kai Chuang
Keyword(s):  
Leucine Zipper ◽  
Infectious Virus ◽  
Repeat Sequence ◽  
Heptad Repeat ◽  
Virus Infectivity ◽  
Gene Products ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associatedgag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.

scholarly journals 5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

2021 ◽  
Vol 118 (37) ◽  
pp. e2112475118
Author(s):  
Pengfei Ding ◽  
Siarhei Kharytonchyk ◽  
Nansen Kuo ◽  
Emily Cannistraci ◽  
Hana Flores ◽  
...  
Keyword(s):  
Rna Processing ◽  
Binding Sites ◽  
Rna Structure ◽  
Virus Assembly ◽  
Wild Type ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Rna Dimerization ◽  
Gag Polyprotein ◽  
Hiv 1

HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. ΨCES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to ΨCES to enable cotranscriptional shedding of the 5′ cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.

scholarly journals Attenuated and Wild-Type HIV-1 Infections and Long Terminal Repeat-Mediated Gene Expression from Plasmids Delivered by Gene Gun to Human Skin ex Vivo and Macaques in Vivo

Virology ◽  
2001 ◽  
Vol 287 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Stephen J. Kent ◽  
Paul U. Cameron ◽  
Jeanette C. Reece ◽  
Phillip R. Thompson ◽  
Damian F.J. Purcell
Keyword(s):  
Gene Expression ◽  
Long Terminal Repeat ◽  
Human Skin ◽  
Ex Vivo ◽  
Terminal Repeat ◽  
Gene Gun ◽  
Wild Type ◽  
Hiv 1

scholarly journals Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 2499-2508 ◽  
Author(s):  
Catherine Ulich ◽  
Amanda Dunne ◽  
Emma Parry ◽  
C. William Hooker ◽  
Richard B. Gaynor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with atat gene deletion (Δtat). Cell lines that contained Δtat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3 Lys were seen in the HIV-1 Δtat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functionaltat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

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