Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

doi:10.1038/s41598-019-46082-2

. 2019 Jul 8;9(1):9806.

doi: 10.1038/s41598-019-46082-2.

Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

Masayuki Amano^{1

2

3}, Haydar Bulut², Sadahiro Tamiya^{1

2}, Tomofumi Nakamura¹, Yasuhiro Koh¹, Hiroaki Mitsuya^{4

5

6}

Affiliations

¹ Department of Hematology, Rheumatology, and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan.
² Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
³ Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo, 162-8655, Japan.
⁴ Department of Hematology, Rheumatology, and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan. mitsuyah@nih.gov.
⁵ Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. mitsuyah@nih.gov.
⁶ Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo, 162-8655, Japan. mitsuyah@nih.gov.

PMID: 31285456
PMCID: PMC6614453
DOI: 10.1038/s41598-019-46082-2

Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

Masayuki Amano et al. Sci Rep. 2019.

. 2019 Jul 8;9(1):9806.

doi: 10.1038/s41598-019-46082-2.

Authors

Masayuki Amano^{1

2

3}, Haydar Bulut², Sadahiro Tamiya^{1

2}, Tomofumi Nakamura¹, Yasuhiro Koh¹, Hiroaki Mitsuya^{4

5

6}

Affiliations

¹ Department of Hematology, Rheumatology, and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan.
² Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
³ Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo, 162-8655, Japan.
⁴ Department of Hematology, Rheumatology, and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan. mitsuyah@nih.gov.
⁵ Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. mitsuyah@nih.gov.
⁶ Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo, 162-8655, Japan. mitsuyah@nih.gov.

PMID: 31285456
PMCID: PMC6614453
DOI: 10.1038/s41598-019-46082-2

Abstract

Accumulation of amino acid (AA) insertions/substitutions are observed in the Gag-protein of HIV-1 variants resistant to HIV-1 protease inhibitors. Here, we found that HIV-1 carrying AA insertions in capsid protein (CA) undergoes aberrant CA degradation. When we generated recombinant HIV-1s (rHIV-1s) containing 19-AAs in Gag, such insertions caused significant CA degradation, which initiated in CA's C-terminal. Such rHIV-1s had remarkable morphological abnormality, decreased infectivity, and no replicative ability, which correlated with levels of CA degradation. The CA degradation observed was energy-independent and had no association with cellular/viral proteolytic mechanisms, suggesting that the CA degradation occurs due to conformational/structural incompatibility caused by the 19-AA insertions. The incorporation of degradation-prone CA into the wild-type CA resulted in significant disruption of replication competence in "chimeric" virions. The data should allow better understanding of the dynamics and mechanisms of CA decomposition/degradation and retroviral uncoating, which may lead to new approach for antiretroviral modalities.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
CA degradation observed in HIV-1 variants with the AA insertion in Gag region. **(a)** Locations of each 19-AA insertion in CA of HIV^TP clones generated by using the Tn5-based transposon system are shown. The sequence of wild-type CA (CA^WT) is shown with its secondary structure diagram. HIV-1 CA consists of the N-terminal domain (NTD; residues 1–145) and the C-terminal domain (CTD; residues 151–231) connected by 5 AAs that act as a flexible linker. Each insertion site is indicated by yellow arrow. The hexamer structure is stabilized by interactions formed by s hairpin and first 3 helices H1, H2, H3 as shown in blue. Two- and three-fold interactions take place between hexamer units are shown in green and orange, respectively. Cyclophilin A-binding site is indicated in purple. **(b)** CA degradation in the lysates of COS-7 cells producing various 19-AAs-containing HIV clones (HIV^TPs). The lysates of COS-7 cells producing HIV^WT or HIV^TPs were examined for enhanced degradation. Each lysate sample was prepared 72 hrs after transfection and subjected to WB with anti-p24 polyclonal anti-serum. The boxes indicate lower-molecular-weight CA degradation products than the mature p24/p25 (CA). Results shown is representative of more than ten independent experiments. **(c)** The CA degradation products are identified in the lysates of HIV_CA^I2-V3. The supernatants of COS-7 cells producing HIV^WT or HIV_CA^I2-V3 were collected 72 hrs after transfection, cleaned with 0.22 μm pore size filters, and subjected to ultracentrifugation (20,000 g, 4 °C, 24 hrs). The obtained virions were lysed and was subjected to WB. Results shown is representative of three independent experiments. **(d)** The lysates of HEK-293 cells producing HIV^WT or HIV^TPs showed the similar degradation products as seen in the COS-7 cell lysates as above. All samples in (b–d) were normalized by their protein concentrations (20 μg/well). Results shown is representative of seven independent experiments. Full-length blots/gels are presented in Fig. S9.

**Figure 2**
Time-dependent progression of the CA degradation in HIV^TPs and recombinant CA-containing 19-AA insert. **(a)** Cell lysates of HIV^WT- or various HIV^TP-producing COS-7 cells were incubated at 37 °C for different periods of time, and the CA concentration of each sample was determined using ELISA. The sample at time 0 was stored at −80 °C immediately following the cell lysate preparation. “Percent p24 (CA)” was determined using equation described in the Methods section. Note that the lysates of COS-7 cells producing a panel of HIV-1-containing the 19-AAs in CA-NTD most rapidly lost CA, followed by the cells producing HIV-1 with the insert in CA-CTD. The data shown represent mean values derived from the results of two independent experiments. **(b)** Morphology of HIV_CA^I2-V3 and HIV_CA^R18-T19. HIV^WT, HIV_CA^I2-V3 and HIV_CA^R18-T19 producing transfected COS-7 cells were subjected TEM study, as described in the Methods section. Note that HIV^WT virions had the typically-condensed mature core, while the cores within HIV_CA^I2-V3 and HIV_CA^R18-T19 are much less condensed or absent. The frequency of mature and aberrant cores are 75.7% (56/74) and 24.3% (18/74) for HIV^WT virions; 25.2% (27/107) and 74.8% (80/107) for HIV_CA^I2-V3 virions; 8.7% (2/23) and 91.3% (21/23) for HIV_CA^R18-T19 virions, respectively. Scale bars: 100 nm. **(c)** Various recombinant CAs such as wild-type HIV-1 CA (rCA^WT) and the 19-AAs-containing CAs (rCA^I2-V3, rCA^R18-T19, rCA^D152-I153, and rCA^G220-V221) were expressed in COS-7 cells, the lysates of the cells incubated at 37 °C, subjected to WB using anti-p24 anti-serum, and the signal densities of rCAs quantified using the NIH Image J Program. Percent p24 (CA) was determined using equation described in the Methods section. The unincubated samples (0 hr) were normalized by their p24 concentrations (5 ng/well). 24 hrs and 48 hrs incubated samples were applied as same volumes as each unincubated (0 hr) sample. Results shown is representative of two independent experiments. **(d)** The same samples as in panel c were subjected to ELISA. Note that all the 19-AAs-containing CAs examined rapidly lost the immunogenicity as compared with rCA^WT. Results shown is representative of two independent experiments. Full-length blots/gels are presented in Fig. S9.

**Figure 3**
The CA degradation observed has no association with cellular/viral proteolytic mechanisms. **(a–d)** MG-132, an inhibitor of proteasome; 3-MeA, an autophagy inhibitor; a mixture of 7 potent cellular protease inhibitors; and SQV, a potent HIV-1-derived aspartyl protease failed to block CA degradation. **(a)** The lysates of COS-7 cells producing HIV^WT or HIV^TPs were prepared 72 hrs after transfection and subjected to WB with anti-p24 polyclonal antibody. MG-132 was added to transfection medium with 20 μM concentration at 6 hrs before harvest. Results shown is representative of four independent experiments. **(b)** The lysates of HEK-293 cells producing HIV^WT or HIV^TP_CA^I2-V3 were prepared 72 hrs after transfection and subjected to WB with anti-p24 polyclonal antibody. HEK-293 cells were incubated for 5 hrs with 5 mM of 3-MeA and were washed just before transfection (lane 2) or were incubated with 3-MeA for 72 hrs after transfection (lane 3). Results shown is representative of two independent experiments. All samples in **(a)** or **(b)** were normalized by their protein concentrations, 30 or 20 μg/well, respectively. **(c,d)** The lysates of COS-7 cells producing HIV^WT or HIV^TP_CA^I2-V3 were prepared 72 hrs after transfection with lysis buffer-containing the PI cocktail or 1 μM of SQV. Data are represented as mean ± S.D. of two independent experiments. **(e)** The lysates of COS-7 cells producing HIV^WT or HIV_CA^I2-V3 were prepared with lysing buffer-containing 13.4 mM of sodium azide, incubated at 37 °C for different periods of time indicated and subjected to ELISA. Note that the presence of a high concentration of sodium azide did not block the CA degradation, suggesting that the degradation event is ATP-independent process. Data are represented as mean ± S.D. of two independent experiments. **(f)** rCA^WT and rCA^R18-T19 produced by *E. coli* staining with Coomassie Brilliant Blue (CBB) are shown. rCA^WT and rCA^R18-T19 were expressed in *E. coli* and each cultured cell preparation was lysed and ammonium-sulfate-precipitated. Subsequently, the lysed samples and ammonium-sulfate-precipitates were subjected to SDS-PAGE and CBB staining. **(g)** The ammonium-sulfate-precipitates of the rCA^WT and rCA^R18-T19 samples described above were run through size-exclusion-chromatography and the fractions-containing rCA^WT and rCA^R18-T19 were subjected to SDS-PAGE and CBB staining. Results shown is representative of two independent experiments. Full-length blots/gels are presented in Fig. S9.

**Figure 4**
The degradation of mutant CAs requires the CA’s C-terminal cleavage and originates from the CA-CTD. **(a)** A genetic map of the three following HIV_CA^I2-V3-based constructs lacking the cleavage(s) of Gag polyproteins generated: (i) a construct, in which the MA-CA cleavage of HIV_CA^I2-V3 was blocked by changing the cleavage site “NYPI” to “NAAI” (thus referred to ^NAAIHIV_CA^I2-V3), (ii) a construct, in which the CA-p2 and p2-NC cleavages were blocked by changing the two cleavage sites “VLAE” and “IMIQ” to “VAAE” and “IAAQ”, respectively (^VAAE/IAAQHIV_CA^I2-V3), and (iii) a construct, in which all the three cleavages were blocked (^{NAAI/VAAE/IAAQ}HIV_CA^I2-V3). **(b)** Reduced degradation in ^VAAE/IAAQHIV_CA^I2-V3 and ^{NAAI/VAAE/IAAQ}HIV_CA^I2-V3. The lysates of COS-7 cells producing HIV_CA^I2-V3 (Lane 1), ^NAAIHIV_CA^I2-V3 (Lane 2), ^VAAE/IAAQHIV_CA^I2-V3 (Lane 3), or ^{NAAI/VAAE/IAAQ}HIV_CA^I2-V3 (Lane 4) were subjected to WB using anti-p24 anti-serum. CA degradation was clearly seen in Lanes 1 and 2, while the degradation levels in Lanes 3 and 4 were drastically reduced, indicating that the blockade of cleavages in the CA-p2-NC cleavage sites resulted in reduction of the CA degradation. **(c)** The CA^I2-V3 degradation products are derived from the CTD of CA^I2-V3. The lysates of HEK-293 cells producing HIV_CA^I2-V3 were subjected to WB using two monoclonal antibodies specific to the CA-NTD (mAb-NTD1 and mAb-NTD2), and two monoclonal antibodies specific to the CA-CTD (mAb-CTD1 and mAb-CTD2). The locations of each immunogen recognized are shown in the top of Panel c. Note that the ladder-like signals were identified only in the lysates examined with the two CA-CTD-specific mAbs. All samples in **(b,c)** were normalized by their protein concentrations (20 μg/well). Results shown is representative of three to five independent experiments for each mAb. Full-length blots/gels are presented in Fig. S9.

**Figure 5**
Degradation-prone HIV^TP-CA^INS variants are replication-incompetent. **(a)** The infectivity of each of HIV^TP-CA^INSNTDs (shown in red) and HIV^TP-CA^INSCTDs (in blue) was determined with the single-round infectivity assay using U373-Magi^CD4+CXCR4+ cells. Note that the infectivity of HIV^TP-CA^INSs was substantially reduced, while that of three HIV^TP-MA^INSs (HIV_MA^V35-W36, HIV_MA^G62-Q63, and HIV_MA^V88-H89) were also substantially reduced. Data are represented as mean ± S.D. (n = 3). **(b)** Loss of replication capacity of HIV^TP-CA^INSNTDs (in red) and HIV^TP-CA^INSCTDs (in blue). Replication kinetics of HIV^TP-CA^INSs was determined with the amounts of p24 production in cultures of MT-4 cells exposed to each HIV^TP. The boxes denote that all HIV^TP-CA^INSs examined completely failed to replicate in MT-4 cells, while the three HIV^TP-MA^INS (in green) described above and HIV_p2^A1-E2 (in purple) also failed to replicate. HIV_p6^T8-A9 (in orange) showed slower replication in MT-4 cells compared to HIV^WT. Data are represented as mean ± S.D. (n = 2).

**Figure 6**
Co-expression of pHIV^WT and pHIV^TP-CA^INSNTD produces highly compromised or replication-incompetent HIV-1. **(a)** HEK-293 cells were transfected with pHIV^WT alone, pHIV^WT plus pHIV^WT, pHIV_MA^E105-E106, pHIV_MA^Q127-V128, pHIV_CA^I2-V3, pHIV_CA^R18-T19, pHIV_CA^A64-A65 or pHIV_p2^A1-E2, cultured for 72 hrs, and the p24 amounts of each culture supernatant were determined. Note that when co-transfected with pHIV^WT and either of 3 pHIV^TP-CA^INSNTD (pHIV_CA^I2-V3, pHIV_CA^R18-T19, pHIV_CA^A64-A65) and pHIV_p2^A1-E2, p24 production was drastically reduced as compared when transfected with pHIV^WT and pHIV^WT plus pHIV^WT. **(b)** HEK-293 cells were transfected with pHIV^WT alone, co-transfected with 1:1, 1:1/6, and 1:1/10 ratios of pHIV^WT and pHIV_p17^Q127-V128, pHIV_CA^I2-V3, or pHIV_CA^R18-T19, cultured for 72 hrs, and the p24 amounts of each culture supernatant were determined. Results shown is representative of three independent experiments. **(c)** The supernatants harvested from each of the cultures described in Panel a were subjected to 9-day HIV-1 replication assays using MT-4 cells as target cells, after all the supernatants were normalized to contain an equal amount of p24. Note that the supernatants from transfection with pHIV^WT alone or pHIV^WT plus pHIV^WT, pHIV_p2^A1-E2, pHIV_MA^E105-E106, or pHIV_MA^Q127-V128 produced large amounts of p24 over the 9-day culture period, while those from transfection with pHIV^WT plus either of the three pHIV^TP-CA^INSNTD (pHIV_CA^I2-V3, pHIV_CA^R18-T19, pHIV_CA^A64-A65) produced no or least p24 protein. All p24 values are single-point determinations.

**Figure 7**
Modeling study of the mechanism of the CA degradation. The 19-AA inserts are indicated by red color for both CA^I2-V3 and CA^R18-T19. On the upper panel, the model of CA^I2-V3 in orange are superimposed into the monomer **(a)** and the hexamer **(b–d)** forms of the CA^WT structure (PDB ID: 5HGL). Similarly, lower panel dedicated to the structural comparison of the CA^R18-T19 model colored in purple with the CA^WT monomer **(e)** as well as the hexamer form **(f–h)** structures. Hexamers are shown from top views (b,f), 90°-rotated side views (c,g), and 180°-rotated views (d,h). Note that the presence of 19-AA inserts let to formation of extended loops, which wrap around the CA pore composed of 6 arginine residues (shown in cyan). It is assumed that the extended loops are likely to push each monomer apart and effectively block the formation of physiologic hexamer complexes.

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References

1. Department of Health and Human Services. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. (2106).
1. Yeni PG, et al. Antiretroviral treatment for adult HIV infection in 2002: updated recommendationsof the International AIDS Society-USA Panel. JAMA. 2002;288:222–235. doi: 10.1001/jama.288.2.222. - DOI - PubMed
1. Grabar S, et al. Factors associated with clinical and virological failure in patients receiving a triple therapy including a protease inhibitor. AIDS. 2000;14:141–149. doi: 10.1097/00002030-200001280-00009. - DOI - PubMed
1. Mitsuya, H. & Erickson, J. W. In Textbook Of AIDS Medicine. (eds Merigan, T. C., Bartlet, J. G. & Bolognes, D.), 751–780 (The Williams & Wilkins Co., Baltimore, Md., 1999).
1. Paredes R, et al. Predictors of Virological Success and Ensuing Failure in HIV-Positive Patients Starting Highly Active Antiretroviral Therapy in Europe. Arch. Intern. Med. 2000;160:1123–1132. doi: 10.1001/archinte.160.8.1123. - DOI - PubMed

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[1] Department of Health and Human Services. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. (2106).

[2] Department of Health and Human Services. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. (2106).

[3] Yeni PG, et al. Antiretroviral treatment for adult HIV infection in 2002: updated recommendationsof the International AIDS Society-USA Panel. JAMA. 2002;288:222–235. doi: 10.1001/jama.288.2.222. - DOI - PubMed

[4] Yeni PG, et al. Antiretroviral treatment for adult HIV infection in 2002: updated recommendationsof the International AIDS Society-USA Panel. JAMA. 2002;288:222–235. doi: 10.1001/jama.288.2.222. - DOI - PubMed

[5] Grabar S, et al. Factors associated with clinical and virological failure in patients receiving a triple therapy including a protease inhibitor. AIDS. 2000;14:141–149. doi: 10.1097/00002030-200001280-00009. - DOI - PubMed

[6] Grabar S, et al. Factors associated with clinical and virological failure in patients receiving a triple therapy including a protease inhibitor. AIDS. 2000;14:141–149. doi: 10.1097/00002030-200001280-00009. - DOI - PubMed

[7] Mitsuya, H. & Erickson, J. W. In Textbook Of AIDS Medicine. (eds Merigan, T. C., Bartlet, J. G. & Bolognes, D.), 751–780 (The Williams & Wilkins Co., Baltimore, Md., 1999).

[8] Mitsuya, H. & Erickson, J. W. In Textbook Of AIDS Medicine. (eds Merigan, T. C., Bartlet, J. G. & Bolognes, D.), 751–780 (The Williams & Wilkins Co., Baltimore, Md., 1999).

[9] Paredes R, et al. Predictors of Virological Success and Ensuing Failure in HIV-Positive Patients Starting Highly Active Antiretroviral Therapy in Europe. Arch. Intern. Med. 2000;160:1123–1132. doi: 10.1001/archinte.160.8.1123. - DOI - PubMed

[10] Paredes R, et al. Predictors of Virological Success and Ensuing Failure in HIV-Positive Patients Starting Highly Active Antiretroviral Therapy in Europe. Arch. Intern. Med. 2000;160:1123–1132. doi: 10.1001/archinte.160.8.1123. - DOI - PubMed

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Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

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Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

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