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The N-end rule and retroviral infection: no effect on integrase.

Boso G, Tasaki T, Kwon YT, Somia NV - Virol. J. (2013)

Bottom Line: The infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted.The N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase.The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology and Genetics Graduate Program, Molecular, Cellular, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT

Background: Integration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-end rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-end rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle.

Results: The infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted. HIV-1 integrase mutants of N-terminal amino acids (coding for stabilizing or destabilizing residues) were severely impaired in their infectivity in both human and mouse cells. Quantitative PCR analysis revealed that this inhibition was mainly caused by a defect in reverse transcription. The decreased infectivity was independent of the N-end rule since cells deficient in N-recognins were equally refractory to infection by the integrase mutants. MLV integrase mutants showed no difference in their infectivity or intravirion processing of integrase.

Conclusions: The N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase. The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

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Only some retroviral integrases bear a N-terminal type-2 destabilizing residue. A comparison of the identity and nature of the amino acid at the N-terminus of mature integrase for various retroviruses. Representatives of the family of retroviruses were chosen based on sequence characterization of the cleavage site or the integrase protein from the literature [22,26-35].
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Figure 4: Only some retroviral integrases bear a N-terminal type-2 destabilizing residue. A comparison of the identity and nature of the amino acid at the N-terminus of mature integrase for various retroviruses. Representatives of the family of retroviruses were chosen based on sequence characterization of the cleavage site or the integrase protein from the literature [22,26-35].

Mentions: Similar to HIV-1, proteins of other retroviruses also harbor N-terminal residues that make them susceptible to the N-end rule. For example mature MLV integrase also bears a type 2 destabilizing residue isoleucine making it a potential substrate for the N-end rule pathway [26]. A comparison of different retroviral integrase N-terminal residues revealed that all lentiviruses examined, gammaretroviruses and spumaretroviruses harbor destabilizing residues. However, this is not universal to retroviruses, since alpha, beta, delta and epsilon retroviruses harbor a stabilizing residue (Figure 4). To test whether MLV infection is affected by the impairment of the N-end rule pathway, we infected our N-recognin deficient MEFs using a MLV based EGFP vector pseudotyped with VSVG. In contrast to HIV-1, MLV infectivity was only slightly decreased in UBR1−/− UBR2−/− UBR4KD cells compared to the other cells tested (Figure 5). Notably the slight decrease was also present in the control UBR1 −/−, UBR2 −/−, Luciferase RNAi cell line. This leads us to conclude that the impact of the N-end rule on MLV infection is minimal. This also allows us to conclude that the decrease observed for HIV-1 on the triply deficient cells is not due to an entry defect since the MLV and HIV vectors are pseudotyped with VSVG and hence utilize the same mode of entry.


The N-end rule and retroviral infection: no effect on integrase.

Boso G, Tasaki T, Kwon YT, Somia NV - Virol. J. (2013)

Only some retroviral integrases bear a N-terminal type-2 destabilizing residue. A comparison of the identity and nature of the amino acid at the N-terminus of mature integrase for various retroviruses. Representatives of the family of retroviruses were chosen based on sequence characterization of the cleavage site or the integrase protein from the literature [22,26-35].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716682&req=5

Figure 4: Only some retroviral integrases bear a N-terminal type-2 destabilizing residue. A comparison of the identity and nature of the amino acid at the N-terminus of mature integrase for various retroviruses. Representatives of the family of retroviruses were chosen based on sequence characterization of the cleavage site or the integrase protein from the literature [22,26-35].
Mentions: Similar to HIV-1, proteins of other retroviruses also harbor N-terminal residues that make them susceptible to the N-end rule. For example mature MLV integrase also bears a type 2 destabilizing residue isoleucine making it a potential substrate for the N-end rule pathway [26]. A comparison of different retroviral integrase N-terminal residues revealed that all lentiviruses examined, gammaretroviruses and spumaretroviruses harbor destabilizing residues. However, this is not universal to retroviruses, since alpha, beta, delta and epsilon retroviruses harbor a stabilizing residue (Figure 4). To test whether MLV infection is affected by the impairment of the N-end rule pathway, we infected our N-recognin deficient MEFs using a MLV based EGFP vector pseudotyped with VSVG. In contrast to HIV-1, MLV infectivity was only slightly decreased in UBR1−/− UBR2−/− UBR4KD cells compared to the other cells tested (Figure 5). Notably the slight decrease was also present in the control UBR1 −/−, UBR2 −/−, Luciferase RNAi cell line. This leads us to conclude that the impact of the N-end rule on MLV infection is minimal. This also allows us to conclude that the decrease observed for HIV-1 on the triply deficient cells is not due to an entry defect since the MLV and HIV vectors are pseudotyped with VSVG and hence utilize the same mode of entry.

Bottom Line: The infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted.The N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase.The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology and Genetics Graduate Program, Molecular, Cellular, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT

Background: Integration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-end rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-end rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-end rule pathway in the HIV-1 life cycle.

Results: The infectivity of HIV-1 but not MLV was decreased in N-recognin deficient cells in which three N-recognins (UBR1, UBR2 and UBR4) were depleted. HIV-1 integrase mutants of N-terminal amino acids (coding for stabilizing or destabilizing residues) were severely impaired in their infectivity in both human and mouse cells. Quantitative PCR analysis revealed that this inhibition was mainly caused by a defect in reverse transcription. The decreased infectivity was independent of the N-end rule since cells deficient in N-recognins were equally refractory to infection by the integrase mutants. MLV integrase mutants showed no difference in their infectivity or intravirion processing of integrase.

Conclusions: The N-end rule pathway impacts the early phase of the HIV-1 life cycle; however this effect is not the result of the direct action of the N-end rule pathway on the viral integrase. The N-terminal amino acid residue of integrase is highly conserved and cannot be altered without causing a substantial decrease in viral infectivity.

Show MeSH
Related in: MedlinePlus