A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

Chiang, Chien Cheng; Wang, Shiu Mei; Pan, Yen Yu; Huang, Kuo Jung; Wang, Chin Tien

doi:10.1128/jvi.01532-09

Cited by 15 publications

(15 citation statements)

References 50 publications

(50 reference statements)

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“…Class II IN mutants do not affect integrase activity but do affect other stages of viral replication, including assembly (14,21,44,45). RT mutants have been shown to significantly reduce virion release (8,9). Furthermore, our results indicate that when IN is removed from Gag-Pol, there is no effect on the cytoplasmic distribution of Gag even in the presence of S6.…”

Section: Discussionsupporting

confidence: 47%

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Cano

Kalpana

2011

J Virol

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INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions.A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55 gag ) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding.

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Section: Discussionsupporting

confidence: 47%

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Cano

Kalpana

2011

J Virol

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“…Samples were subjected to 8 or 10 % SDS-PAGE and electroblotted onto nitrocellulose membranes. Protocols for detection of membrane-bound proteins were as described previously (Chiang et al, 2010) using an enhanced chemiluminescence (ECL) detection system according to the manufacturer's protocol (Pierce). HIV-1 Gag/Gag-Pol or HA-tagged NEDD4L proteins were probed by using an anti-p24gag (mouse hybridoma clone 183-H12-5C) or anti-HA (LTK BioLaboratories) mAb.…”

Section: Methodsmentioning

confidence: 99%

p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

Guo

Huang³

et al. 2016

Journal of General Virology

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During virus assembly, HIV-1 Gag-Pol is packaged into virions via interaction with Pr55gag. Studies suggest that Gag-Pol by itself is incapable of virus particle assembly or cell release, perhaps due to the lack of a budding domain in the form of p6gag, which is truncated within Gag-Pol because of a ribosomal frameshift during Gag translation. Additionally (or alternatively), large molecular size may not support Gag-Pol assembly into virus-like particles (VLPs) or release from cells. To test these hypotheses, we constructed Gag-Pol expression vectors retaining and lacking p6gag, and then reduced Gag-Pol molecular size by removing various lengths of the Pol sequence. Results indicate that Gag-Pol constructs retaining p6gag were capable of forming VLPs with a WT HIV-1 particle density. Gag-Pol molecular size reduction via partial removal of the Pol sequence mitigated the Gag-Pol assembly defect to a moderate degree. Our results suggest that the Gag-Pol assembly and budding defects are largely due to a lack of p6gag, but also in part due to size limitation.

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“…Several groups have introduced amino acid substitutions at different subdomains of RT (11,31,33,(41)(42)(43)(44)(45). Many of these mutations led to destabilization of the RT protein in the virus particle as well as a loss of infectivity.…”

Section: Figmentioning

confidence: 99%

The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles

Boso

Örvell

Somia

2015

J Virol

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Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits. IMPORTANCEHIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process. Like all retroviruses, human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, synthesizes and packages its main structural and enzymatic proteins as precursor polyproteins. For HIV-1, these polyproteins are p55 (Gag) and p160 (GagPol). Gag is the most abundant polyprotein and is translated from a genome-length mRNA that contains the Gag and GagPol open reading frames. The synthesis of GagPol requires a ribosomal frameshift leading to a Gag/GagPol ratio of about 20:1 in the virus particle (1). Individual mature viral proteins are generated following viral assembly as a result of a series of proteolytic cleavage events at specific positions catalyzed by the viral protease, which is synthesized as a part of GagPol (2).One protein that is released as a result of proteolytic processing of GagPol is reverse transcriptase (RT). RT catalyzes the reaction for the conversion of viral RNA to...

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A Single Amino Acid Substitution in HIV-1 Reverse Transcriptase Significantly Reduces Virion Release

Cited by 15 publications

References 50 publications

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6

p6gag domain confers cis HIV-1 Gag-Pol assembly and release capability

The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles

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