aCentro Nacional de Biología Fundamental, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain; bComplejo Hospitalario Universitario de Santiago de Compostela; 15705 Santiago de Compostela, La Coruña, Spain; and cConsellería de Sanidade e Servicios Sociais, Dirección Xeral de Saúde Pública, Xunta de Galicia, Spain.
Sponsorship: This work was partly funded by grants MBVI 1023/99/2 and SBVI 1090/00/01 from Plan Nacional del SIDA, Ministerio de Sanidad y Consumo, Spain, and by Scientific Agreement with the Government of Galicia, Xunta de Galicia, Spain.
Received: 21 September 2001;
revised: 12 October 2001; accepted: 16 October 2001.
The HIV-1 epidemic in western Europe is dominated by B-subtype viruses, but non-B and intersubtype recombinants are being identified with increasing frequency . Little is known about the biological characteristics of the recombinant viruses and the relationship with their transmissibility and clinical progression [2,3]. The V3 loop of B-subtype viruses has been shown to play a role in the syncytium-inducing (SI) phenotype, viral tropism and in the differential usage of the chemokine receptors (CCR5, CXCR4) . Although all subtypes can use co-receptors CXCR4 or CCR5, some preferences relative to subtypes have been described [5–8].
In general, non-SI variants display low V3 net charge (≤ +4), whereas SI variants emerge late in the disease course and display higher V3 net charges (≥ +5) [9,10].
In order to determine the biological characteristics of the newly described BG recombinants [11–13] we sequenced the V3 loop from plasma RNA of eight BG strains and eight B-subtype viruses as controls, obtained from HIV-1-infected patients who were attending the same hospitals at the time the study was carried out. Moreover, recombinant BG virus was isolated from the peripheral blood mononuclear cells (PBMC) of one patient and its in-vitro biological properties were studied, including the cell tropism, replication capacity, SI and co-receptor usage.
Epidemiological data are shown in Table 1. All patients were native Spanish, except one from Cabo Verde (X-623). The V3 net charge was calculated on the basis of the difference between the number of positively charged amino acid residues, arginine (R) or lysine (K), and the number of negatively charged residues, aspartic acid (D) or glutamic acid (E) . The prediction of SI phenotype and CXCR4 or CCR5 co-receptor usage was made considering the net charge and by analysing the presence of positively charged amino acids within specific positions that have previously been shown to influence the biological in-vitro phenotype: 306, 320 and 324 positions [15,16].
Virus isolation and biological characterization from PBMC were performed as previously described . Co-receptor usage was determined by infecting, with cell-free viral stock, the human glioma cell line, U87.CD4, stably expressing a chemokine receptor CCR1, CCR2b, CCR3, CCR5 or CXCR4, and the human osteosarcoma cell line, GHOST, expressing either CCR3, CCR5, CXCR4, BOB/GPR15 or BONZO(STRL33) [18,19]. Cell cultures were observed daily for cytopathic effects, and supernatant was harvested on day 7 for the detection of p24 antigen (Innotest HIV antigen monoclonal antibody; Innogenetics, Zwijndrecht, Belgium). GHOST cells were analysed using a FACScan flow cytometer. The V3 region was sequenced from patient PBMC proviral DNA and from in-vitro HIV-1 primary isolate.
V3 sequences are shown in Table 1. The estimated V3 net charge was +5.1 (with a range of +4 to +6) for BG recombinant strains, and +4 (with a range of +3 to +5) for B subtypes. The presence of a basic amino acid (R) at the 306 phenotype predictive position was observed in three BG recombinants and in three B-subtype V3 sequences. Other basic amino acid substitutions at phenotype-predictive positions were not detected. However, the net charge in the majority of the BG recombinants was predictive of SI/CXCR4, but they lacked phenotype-associated signature amino acids. Similar findings have been described in some subtype-F isolates , suggesting that multiple basic substitutions within the V3 loop, associated with an overall net charge, may be a general requirement for generating the SI/CXCR4 phenotype. It should be noted that most BG recombinant sequences showed several characteristic mutations, corresponding to: 308T, 309M, 314V, 315L, 320Q, 327K (Table 1). These changes were not detected in the B-subtype viruses analysed in this study. Moreover, the frequency of these mutations in the B-subtype sequences published in the Los Alamos Data Base is low, being observed at a range of 5–16%. An important aspect will be to determine whether these divergences between the BG recombinant strains and B-subtype viruses will result in biological differences.
One BG recombinant primary isolate (X-421) was obtained from a patient in A1 clinical stage. Its biological characteristics were: rapid/high replication, SI phenotype, and the usage of CCR2b, CCR3, CCR5 and CXCR4 co-receptors. A V3 amino acid sequence, corresponding to proviral DNA obtained from patient PBMC, was compared with those obtained from primary isolate and MT2 cell lysates. The sequences derived from in-vitro samples showed a positively charged amino acid (K) at the 320 position, resulting in a net charge of +7. This mutation was not observed either in proviral DNA from patient PBMC or in plasma RNA, indicating its emergence under culture-selective pressure.
We have described, for the first time, the biological characteristics of the BG recombinant primary isolate. Interestingly, the SI/X4 phenotype was obtained from a patient in the early clinical stage. The follow-up of this patient will allow us to establish whether these findings are associated with a faster progression of the disease.
The Spanish Group for Antiretroviral Resistance Studies in Galicia
A. Agulla, A. Mariño, Hospital Arquitecto Marcide, Ferrol (La Coruña); S. López-Calvo, J.D. Pedreira, Hospital Juan Canalejo (La Coruña); A. Aguilera, E. Losada, A. Prieto, Complejo Hospitalario Universitario de Santiago (La Coruña); J. Corredoira, M.J. López- Alvarez, A. Rodríguez, Hospital Xeral-Calde (Lugo); M. Bustillo, J. García-Costa, R. Fernández-Rodríguez, Hospital Nuestra Señora del Cristal (Orense); R. Rodríguez, Hospital Provincial Santa María Madre (Orense); C. Miralles, A. Ocampo, Hospital Xeral-Cíes, Vigo (Pontevedra); R. Ojea de Castro, Hospital Montecelo (Pontevedra); L.E. Morano, R. Pérez-Rodríguez, A. Rodríguez, J. Torres, Hospital Meixoeiro, Vigo (Pontevedra); J. Díz, R. Rodríguez.-Real, Hospital Xeral Provincial (Pontevedra).
María Luisa Villahermosaa
María Teresa Cuevasa
Elena Vázquez de Pargaa
Michael M. Thomsona
José A. Taboadac
and the Spanish Group for Antiretroviral Resistance Studies in Galicia
The authors would like to thank Dr José María Hernández Cochón, Conselleiro de Sanidade e Servicios Sociais, and Dra Pilar Farjas Abadía, Directora Xeral de Saúde Pública, Consellería de Sanidade e Servicios Socias, Xunta de Galicia for their support in the development of the study in Galicia. The technical assistance of Milagros Pinilla and Concepción González-Troncoso is gratefully acknowledged.
1. McCutchan FE. Understanding the genetic diversity of HIV-1. AIDS 2000, 14: S31–S44.
2. Leitner T, Escanilla D, Marquina S. et al
. Biological and molecular characterization of subtype D, G, and A/D recombinant HIV-1 transmission in Sweden. Virology 1995, 209: 136–146.
3. Kusagawa S, Takebe Y, Yang R. et al
. Isolation and characterization of a full-length molecular DNA clone of Ghanaian type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range. AIDS Res Hum Retroviruses 2001, 17: 649–655.
4. Distler O, McQueen PW, Tsang ML. et al
. Primary structure of the V3 region of gp120 from sequential human immunodeficiency virus type 1 isolates obtained from patients from the time of seroconversion. J Infect Dis 1995, 172: 1384–1387.
5. Tscherning-Casper C, Vodros D, Menu E. et al
. Coreceptor usage of HIV-1 isolates representing different genetic subtypes obtained from pregnant Cameroonian women. European Network for in Utero Transmission of HIV-1.
J Acquir Immune Defic Syndr 2000, 24: 1–9.
6. Abebe A, Demissie D, Goudsmit J. et al
. HIV-1 subtype C syncytium- and non-syncytium-inducing phenotypes and coreceptor usage among Ethiopian patients with AIDS. AIDS 1999, 13: 1305–1311.
7. Kato K, Sato H, Lakebe Y. Role of naturally occurring basic amino acid substitutions in the human immunodeficiency virus type 1 subtype E envelope V3 loop on viral coreceptor usage and cell tropism. J Virol 1999, 73: 5520–5526.
8. Berger EA, Doms RW, Fenyo EM. et al
. A new classification for HIV-1. Nature 1998, 391: 240.240.
9. Bratt G, Leandersson AC, Albert J. et al
. MT-2 tropism and CCR5 genotype strongly influence disease progression in HIV-1 infected individuals. AIDS 1998, 12: 729–736.
10. Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change in coreceptor use correlates with disease progression in HIV-1 infected individuals. J Exp Med 1997, 185: 621–628.
11. Thomson MM, Delgado E, Manjón N. et al
. HIV-1 genetic diversity in Galicia Spain: BG intersubtype recombinant viruses circulating among injecting drug users. AIDS 2001, 15: 509–516.
12. Pérez Alvarez L, Cuevas MT, Villahermosa ML. et al
. Prevalence of drug resistance mutations in B, non-B subtypes, and recombinant forms of human immunodeficiency virus type 1 in infected individuals in Spain (Galicia). J Hum Virol 2001, 4: 35–38.
13. Pérez-Alvarez L, Thomson MM, Villahermosa ML. et al
. HIV-1 subtype G and BG recombinant viruses in Spanish natives: evidence of characteristic mutations in reverse transcriptase and protease. AIDS 2001, 15: 1907–1510.
14. Briggs DR, Tuttle DL, Sleasman JW, Goodenow MM. Envelope V3 amino acid sequence predicts HIV-1 phenotype (co-receptor usage and tropism for macrophages). AIDS 2000, 14: 2937–2955.
15. Fouchier RAM, Groenink M, Koostra NA. et al
. Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp 120 molecule. J Virol 1992, 66: 6777–6780.
16. de Jong J-J, de Ronde A, Keulen W. et al
. Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J Virol 1992, 66: 6777–6780.
17. Pérez Alvarez L, Verdejo J, González Lahoz J. et al
. Expresión de diferentes subpoblaciones fenotípicas del VIH-1 a lo largo de la historia natural de la infección. Medicina Clínica 1998, 110: 441–445.
18. Bjorndal A, Deng H, Jansson M. et al
. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 1997, 71: 7478–7487.
19. Cecilia D, Kewalramani VN, O'Leary J. et al
. Neutralization profiles of primary human immunodeficiency virus type 1 isolates in the context of coreceptor usage. J Virol 1998, 72: 6988–6996.
20. Holm-Hansen C, Baan E, Asjo B. et al
. Determinanats for syncytium-inducing phenotype of HIV-1 subtype F isolates are located in the V3 region. AIDS Res Hum Retroviruses 2000, 16: 867–870.