Biomedical Science

Original Paper

J Biomed Sci 2000;7:128-135 Received: September 10, 1999 Accepted: October 21, 1999

Rapid Full-Length Genomic Sequencing of Two Cytopathically Heterogeneous Australian Primary HIV-1 Isolates

Rober t B. Oel r ichs a V ic to r ia A. Lawson b,c Karen M. Coates b,c

Cather ine Chat f ie ld b N icho las J. Deacon a Da leA . McPhee b,c

AIDS aMolecular and bCellular Bio logy Units, Macfartane Burnet Centre for Medical Research, Fairfield, and cDepartment of Microb io logy and Immunology , Universi ty of Melbourne, Parkville, Vic., Austral ia

Key Words HIV, rapid sequencing, full-length • HIV, Australian isolates

Abstract Two Australian HIV-1 isolates, derived from patient blood (HtVMBc200) and cerebrospinal fluid (HIVMBc925), were characterized after in vitro culture in peripheral blood mononuclear cells (PBMC). Although virus replica- tion was similar, as measured by cell-free reverse trans- criptase activity, only one of the two isolates (HIV- 1MCB200) consistently induced cell syncytia and depleted the PBMC population of CD4+ cells by cell killing. A novel technique, devised for rapidly obtaining high-quality vi- ral sequence data and the full-length genomic sequence of these two isolates, is presented. Analysis of the pre- dicted sequence of the viral Env proteins provides corre- lates of the observed phenotypes. Phylogenetic analysis derived using near full-length sequence of these and oth- er HIV-1 subtype B genomic sequences (including two other Australian isolates) shows a star-shaped phyloge- ny with each member having a similar genetic diversity. These data expand the database of genomic sequence available from well-characterized primary clinical iso- lates of HIV-1 using a novel rapid technique.

Copyright @ 2000 National Science Council, ROC and S. Karger AG, Basel

KARG E IK Fax+41 61 306 12 34 E-Mail karger@karger, ch www. karger.com

© 2000 National Science Council, ROC S. Karger AG, Basel 1021-7770/00/0072-0128517.50/0 Accessible online at: www. karger.com/joumals/jbs

Despite the intensive study of HIV- 1 genetics in West- ern countries, only 52 subtype B isolates have been sequenced in their entirety (excluding closely related clones from the same biological source [2 I]). The majority of these represent laboratory-adapted strains that have been multiply passaged in immortal cell lines. Such HIV- 1 strains provide only a limited model of the diversity and adaptive characteristics of viruses present in vivo. More data are required from the characterization of primary clinical isolates, to reveal the determinants of cell tropism and replication kinetics at the level of the entire genome.

More extensive sequence data covering the breadth and evolution of the global HIV-1 epidemic are also urgently required. Of the 75 non-subtype B isolates that have been fully sequenced, 18 have revealed a recombi- nant genomic structure [ 13, 21, 41 ]. Recent data, especial- ly from African isolates [27, 35], and the application of sophisticated phylogenetic tools, are revealing a picture of increasing genomic complexity that will have implica- tions for both molecular epidemiology and future vaccine design.

The HIV-1 epidemic in Western countries has been largely caused by subtype B. This has made molecular epi- demiological studies difficult, due to the similar DNA sequences of the viruses being examined. Such phyloge- netic analyses are considerably enhanced by the analysis

Dr. Robert B. Oetrichs H1V-NAT, Program on AIDS, Thai Red Cross Society 1871 Rama IV Rd. Pathumwan, Bangkok 10330 (Thailand) Tel, +66 2256 4579, Fax +66 2653 2488, E-Mail oelrichs@loxinfo.co,tb

of large continuous stretches of sequence. With the ability to rapidly sequence the entire genome of a particular iso- late, these techniques can be applied to more finely dissect patterns of viral transmission and evolution.

This paper presents the study of two Australian primary clinical HIV- 1 isolates from early in the epidemic. Derived from distinct tissue origins (peripheral blood and cerebro- spinal fluid, CSF), the viruses exhibit strikingly different in vitro characteristics, including CD4+ T-cell depletion, cell killing and co-receptor usage. A method is described whereby high-quality full-length genomic sequence was rapidly derived from these isolates. Phylogenetic analysis was employed to determine the genetic relationships of full-length subtype B genomes, including four Australian isolates, revealing all members analyzed to be nearly equal- ly distinct. It is anticipated that the improved facility of deriving full genomic sequence from given isolates will allow more powerful analyses of the molecular epidemiolo- gy of the Western epidemic, as well as enhancing the pic- ture of HIV-1 recombination and evolution worldwide.

Materials and Methods

Viral Strains and Cell Types Two Australian HIV- 1 isolates, HIV- 1MCB200 (Sydney, March 18,

1986) and HIV-1 MBC925 (Melbourne, May 15, 1987), were isolated as previously described [32]. HIV-1MBC200 was derived by co-ctflture of donor and patient peripheral blood mononuclear cells (PBMC). HIV-IMBc925 was derived from CSF. Both patients had AIDS and T4:T8 ratios of< 0.2 at the time of isolation. Virus used for biological cloning had been passaged only 3 times in donor phytohemagglutinin (PHA)-stimulated PBMC. Biological cloning was achieved by three rounds of limiting dilution.

Co-Receptor Usage Co-receptor usage was determined by the amplification of an

HIV-1 gag PCR product from infected HOS-CD4 cells expressing either CCR1, CCR2b, CCR3, CCR5 or CXCR4. These cells were obtained through the AIDS Research and Reference Reagent Pro- gram, Division of AIDS, NIAID, NIH from Dr. N. Landau. A mono- layer of 1 x 105 HOS cells was inoculated with 1 x 103 TCIDs0 of virus stock, which had been pretreated with 10 U/ml DNase I (Boeh- ringer Mannheim, Germany) for 30 min at room temperature. Four days postinfection the monolayer was washed with PBS and lysed for PCR [28]. An HIV-1 gag-specific PCR product was amplified in a nested reaction containing 0.2 laM of the external primer pair A2/B2 (NL4.3 base position (bp) 1363-1390/1819-1847) [24] or internal primer pair SK38/SK39 (bp 1544-1571/1631-1658) [37], 0.2 mM dNTP and 2.5 U Taq DNA polymerase (Boeringer Mannheim). Reaction conditions were: 2 min at 94 ° C, 30 cycles of 94 ° C for 15 s, 55°C for 15 s and 72°C for 45 s followed by a final extension at 72 °C for 2 min. The 114 nucleotide (nt) product was visualized on a 2% agarose gel. No product was detected in uninfected HOS cells or in the DNase-treated inoculum.

Genomic Sequencing Low-molecular-weight DNA was isolated from PBMC culture

lysates by a modification of the method of Hirt as described [20, 46]. Long-range PCR was performed with an Expand Long Template PCR Kit (Boehringer-Mannheim), using the manufacturer's reagents and conditions and primers MSF1 and MSR5R (table 1), yielding an amplified product between bp 623 and 9629 (NL4.3). This long- range PCR product was used directly as a template for PCR with the primer pairs HIV-B2-B 14 (table 1). Reactions were performed using Taq polymerase (Boehringer-Mannheim) and the manufacturer's supplied reaction buffer with an additional 1 mM MgCI2. The reac- tion conditions were 95 ° C for 2 rain, annealing at 60 ° C and exten- sion at 72 °C for 1 rain for 4 cycles, followed by 37 cycles of denatur- ation at 95 ° C and extension at 72 ° C for 1 rain. For the primer pair HIV-B7 the initial annealing temperature was at 55°C. Products from the primer pair HIV-B1 were generated by a nested PCR using firstly HIV-BtF and HIV-B3R and identical conditions as above with 2-rain extension. This product was used as a template for the HIV-B1 primer pair. HIV-B PCR products were purified using a PCR spin kit (Qiagen, Germany), quantified by UV spectrophotome- try and sequenced using M13 forward and reverse fluorescent- labeled primers with SequiTherm Long Read sequencing kits (Epi- centre Technologies, Wisc., USA) and the LI-COR 4000L automated DNA sequencer and gels read using the BaseImager IR TM software (LI-COR, Nebr., USA). DNA sequence contig assembly and initial analysis was performed using Geneworks version 2.3 (Oxford Molec- ular Group Inc., Calif., USA).

Phylogenetic Analysis All unique HIV-1 subtype B fulMength genomic sequences avail-

able in the HIV sequence database, LOs Alamos National Laboratory, as at October 1998, were truncated at the 5' untranslated region of gag to the end of env (bp 668-8782 in NL4.3). Isolates used were: ACH 1 and ACH2 (accession numbers U34603 and U34604, respec- tively [18]), AD8 (AF004394 [48], BC (L02317 [14]), BRU (K02013 [49]), CAM1 (D10112), DH12 (AF069140 [45]), HAN (U43141 [44]), HXB2 (K03455 [52]), JRCSF and JRFL (M38429 and U63632, respectively [26]), LW12.3 (U12055 [39]), MANC (U23487 [55]), MBC200 (AF042100), MBC925 (AF042t01), MBCD36 (AF042105 [34]), MN (M17449 [19]), NY5 (M38431 [51]), OYI (M26727 [22]), P89.6 (U39362 [6]), PV22 (K02083 [31]), RF (M17451 [47]), RE42 (U71182 [15]), SF2 (K02007 [43]), TH475 (L31963 [33]), VII (AF146728 [36]), WEAU (U21135), WR27 (U26546 [42]), YU2 and YUt0 (M93258 and M93259, respectively [29]). Alignment was by the CLUSTALW algorithm (randomized input of sequences, gap penalty 20) and a genetic distance matrix was generated using EDNADIST, employing the Jukes Cantor model of molecular evolution, correcting for multiple substitutions, with a transition:transversion ratio of 2.0. Phylogeny was inferred by ENEIGHBOR (randomized input of sequences) and the tree built and manipulated using XTREE. All analyses were performed at the Australian National Genomic Information Service, WAG interface.

Full-length sequences from three members of the Sydney Blood Bank Cohort transmission cluster other than D36 [34] were excluded from the analysis. Due to the deletions and rearrangements present in the nef/LTR of isolate D36, this genomic region was excluded from the analysis in all sequences.

Rapid Full-Length Analysis of Australian HIV-1 Isolates

J Biomed Sci 2000;7:128-135 129

Table 1. Nucleotide sequence and properties of PCR primers used in full-length sequencing

Primer . . . . . . . n a m e

MSF1 MSR5R HIVB 1F HIVB1R HIVB2F HIVB2R HIVB3F HIVB3R HIVB4F HIVB4R HIVB5F HIVB5R HIVB6F HIVB6R HIVB7F HIVB7R HIVBSF HI~q~8R HIVB9F HIVB9R HIVB 10F HIVB 1 OR HIVB 11F HIVB 11R HIVB 12F HIVB 12R HIVB 13F HIVB 13R HIVB14F HIVB 14R

~ ~ i ~ ~: i ̧¸ i ¸ ~ : ii ~ Z! i~ ~ i ¸~ ~ ~i ~ ~ ii ~ ; ~

Primer sequence : NL~3

; : : } ; ; ;

ii ii ̧ i ~̧ il ~i ̧ i i ! ~ ~ i ~ Bas~ position: T m Pr imer Product

length, nt length, bp

aaa,tct,ct a,gca,gtg,gcg, ccc,gaa,cag + 623-649 65 ° 27 acg,tgc,cct,caa,ggc,aag,ctt,tat,tga,ggc,t - 9629-9596 64 ° 31 9,006 M 13Ftg,gaa,ggg,cta,att,cac,tca, cgg,aaa,ag + 1-28 65 ° 50 M 13Ra,ccg,acg,ctc,tcg,cac,cca, tc - 810-789 64 ° 44 850 M 13Fcc,cga,aca,ggg,act,tga,aag,c + 641-661 58 ° 43 M 13Rt,ctg,cag,ctt,cct,cat,tga,tgg,tc - 1421 - 1398 60 ° 47 830 M 13Fgg,tca,gcc,aaa,att,acc,cta,tag + 1170-1192 56 ° 45 M 13Rt,gtc,ctt,cct,ttc,cac,att,tcc - 2053-2029 56 ° 45 920 M 13 Fag,cca,taa,agc,aag,agt,ttt,ggc + 1856-1880 57 ° 45 M13Rt,tcc,att,tct,gta,caa,att,tct, act,aat,gc - 2775-2646 57 ° 53 894 M 13Fga,aga,aat,ctg, ttg,act,cag, att,gg + 2509-2534 54 ° 47 MI 3Rt,cat,aat,aca,ctc,cat,gta, c - 3510-3489 53 ° 43 1,040 M 13Fac,tcc,atc,ctg,ata,aat,gga,cag + 3249-3270 54 ° 45 M 13Rt,atc,tgg,ttg,tgc,ttg, aat,gat,tc - 4 t 36-4113 54 ° 47 880 MI 3Faa,tta,gga,aaa,gca,gga,tat,gtt,act,g + 3901-3927 54 ° 49 M 13Ra,gtt,tgt,atg, tct,gtt,gct,att,atg,tct,ac - 4859-4830 53 ° 53 1,000 M 13Fta,tag,gac,agg,taa,gag, atc,agg,ctg + 4712-4737 55 ° 48 M 13Rc,act,agg,caa,agg, tgg,ctt,tat,c - 5536-5514 55 ° 46 870 M13Fcg,aac,tag,cag,acc,aac,taa,ttc + 5339-5362 54 ° 46 M 13Rc,att,gcc,act,gtc,ttc,tgc,tc - 6223-6203 53 ° 44 930 M 13Fct,atg, gca,gga,aga,agc,gga,gac + 5966-5989 62 ° 45 M 13Ra,cca,gcc,ggg,gca,caa,taa,tg - 6880-6860 61 ° 44 980 M 13Fgc,atg,agg, ata,taa, tca,gtt,tat,ggg + 6531-6556 55 ° 48 M 13Rt,ggg,agg,ggc,ata,cat,tgc - 7529-7511 55 ° 47 890 M 13Fag,gag,ggg,acc,cag, aaa,ttg + 7308-7328 58 ° 42 M 13 Rc,aaa,tga,gtt,ttc,cag,agc,aac,cc - 8056-8025 59 ° 47 780 M 13Fgg,gct,att,gag,gcg,caa,c + 7884-7901 54 ° 40 M 13Rc,tat,ctg,tcc,cct,cag, cta,ct g,c - 8697-8675 55 ° 46 850 M 13Ftt,gag,aga,ctt,act,ctt,gat,tgt,aac,g + 8525-855 t 54 ° 49 M13Rt,ccc,agg,ctc,aga,tct,ggt,cta,ac - 9563-9540 53 ° 47 1,080

The position in the HIV-1 genome to which each primer is designed is indicated relative to the bp in NIA.3. In primers containing an M 13 universal sequence tail, the following sequences are indicated by abbreviation: M13F = 19 nt M13 forward universal sequence tgc,cac,gac,gtt,gta,aaa,cga,c; M 13R = 20 nt M 13 reverse universal sequence tgc,gga,taa,caa,ttt,cac,aca,gg.

Results

Viral Replication Kinetics' and Cytopathicity In comparisons of the replication kinetics of HIV-

1 MBC925 and HIV-1MBC200 with several other primary iso- lates (from brain, CSF and peripheral blood) and refer- ence isolates (IIIB, NL4.3, AD8) these two consistently achieved high cell-free reverse transcriptase activity (RT), with almost identical replication kinetics in PBMC using equivalent virus input (multiplicity of infection (MOI) was 0.001; data not shown). Only HIV- 1MBC200 was able to replicate in CEM or MT-2 cell lines (data not shown). In contrast to showing equivalent replication kinetics, HIV-1MBC925 did not cause significant cytopathic effects

in e i t h e r P B M C o r p u r i f i e d C D 4 + T cel ls w h e r e a s b a l l o o n -

ing a n d s y n c y t i u m t b r m a t i o n w e r e c o n s i s t e n t l y o b s e r v e d

fo r H I V - 1 MBC200-infected cel ls ( d a t a n o t s h o w n ) [2 5]. A d d i -

t iona l ly , s u r v i v a l o f C D 4 + T cel ls w a s m a r k e d l y d i f f e r e n t

fo r HIV-1MBC925 c o m p a r e d to HIV-1MBC200 w i t h far f e w e r

cells b e i n g k i l l ed o v e r t he 14-day p e r i o d t e s t ed (fig. 1).

A l t h o u g h C D 4 d o w n - m o d u l a t i o n w a s o b s e r v e d fo r b o t h

v i ru se s ea r ly d u r i n g i n f e c t i o n , i t was C D 4 + T-ce l l loss t ha t

w a s m o s t e x t e n s i v e in HIV-1MBC2o0-infected cel ls a t day 14

p o s t i n f e c t i o n (da t a n o t shown) . S u r v i v a l o f C D 4 + T cel ls

c o r r e l a t e d w i t h t r o p i s m , as d e m o n s t r a t e d by c o m p a r i s o n

w i t h t h e M - t r o p i c a n d T - t r o p i c r e f e r e n c e s t r a ins A D 8 a n d

N L 4 . 3 , r e s p e c t i v e l y (fig. 1). T h e s e d a t a c o n f i r m T - t r o p i s m

o f HIV-1MBC200 a n d M-tropism o f HIV-1MBC925.

130 J Biomed Sci 2000;7:128-135 Oelrichs/Lawson/Coates/Chatfield/Deacon/ McPhee

Fig. 1. Comparison of CD4+ T-cell survival on HIV-1 infection of PHA-activated PBMC. PHA-activated PBMC (1 x 106 cells) were infected with 1,000 TCIDs0 of reference isolates HIV-1NL4.3 (NL4.3), or HIV-1AD8 (ADS) and primary HIV-1MBC200 (200) or HIV-IMBc925 (925). Cells were maintained for 14 days [30] and the viable CD4+ T-cell population analyzed by flow cytometry [16]. Data are compiled from three separate experiments with standard error bars shown.

m

70

60

50

40

30

20

10

0 MockNL4.3 AD8 200

Viral isolate 925

r e v

I gag ] i~ I ta2V .._~___l ]'"nef I

0 1 2 3 4 5 6 7 8 9 i ......... i ~ l z 1 i l ~ I kb

Primary PCR . . . . Products

1 3 5 7 9 11 13 HIV-B PCR

2 ~ " 4 i,llll 6 " ' " " - 8 ~ 1 0 ' ' ' ' ~ 12 " " " ' " - 14 Products

Fig. 2. Diagram of the HIV-1 genome showing major open reading frames. Sequencing proceeds by the generation of a near fulMength PCR product extending from the 5' untranslated region of the gag gene to 3' end of U5 in the 3'LTR. An additional primary PCR product is generated to sequence the 5'LTR. These primary products are used as tem- plates in a series of secondary PCR reactions, using M13-tailed primers based on highly conserved regions of the HIV-1 genome. These secondary PCR reactions generate sufficient template for automated DNA sequencing, data from which are assembled into the final complete sequence.

Second Receptor Usage Isolates HIV-1NL4.3 and HIV-1Mgc200 were able to

infect cells expressing CXCR4 as predicted by their char- acterization as T-cell tropic, SI isolates (data not shown). In addition, they were also able to infect cells expressing CCR1, CCR3 and CCR5. T-tropic or St isolates have been reported to infect HOS.CD4 cells lacking co-recep- tor expression [4, 5] or expressing co-receptors other than CXCR4 [4, 5, 53]. This may reflect the low levels of endogenous CXCR4 on HOS cells detected by RT-PCR [5]. The M-tropic isolates HIV-1AD8 and HIV-1MBC925

were only consistently able to use the CCR5 co-receptor (data not shown). HIV-1ADA or its molecular clone HIV-1AD8 has been shown to utilize CCR5 [5, 9] and to a lesser extent CCR3 [5] in HOS.CD4 cells and other co- receptor expressing celt lines [1, t0].

Genomic Sequencing The long-range PCR technique used enabled the near

full-length genomic sequence of HIV-1 to be accurately determined for each isolate in less than a week (fig. 2). The overall genomic structure of the isolates was normal

Rapid Full-Length Analysis of Australian HIV- 1 Isolates

J Biomed Sci 2000;7:128-135 13t

Fig. 3. Unrooted phylogenetic tree gener- ated by neighbor-joining analysis showing available full-length HIV-1 subtype B ge- nomic sequences. The four Australian iso- lates (MBC200, MBC925, MBCD36 and VH) are italicized. Paired isolates from the same patients (A.C.H., J.R. and Y.U.) and five strains derived from the same source are circled. The primary clinical isolates P89.6 and WR27 are indicated in bold type. The star-shaped phylogeny reflects an equivalent genetic distance between all sequences (apart from the closely related species mentioned) and indicates that the Australian HIV-1 sub- type B epidemic has been contributed to sub- stantially by the introduction of diverse viral strains.

DHI2 ----~-~

OY

SF2

RF ~ J

P89.6

WEAU

/ /

MBCD36

WR27

BC MBC925

NY5 CAM1 RL42

VII J

N

MANC

0.01

MN

with intact open reading frames predicting the protein sequence of Gag, Pol, Env, Vif, Vpr, Vpu, Tat, Rev and NeE Additionally, the structure of the long terminal repeat (LTR) was normal in both isolates, with the identi- fication of intact transcription factor binding sites.

Phylogenetic Analysis Neighbor-joining phylogenetic analysis was performed

using HIV-1 strains representative of the global distribu- tion of subtype B epidemic and the four Australian sub- type B isolates (HIV- 1MBc200, HIV- 1 MBC925, HIV- 1 MBCD36 and HIV-lVH) that have been sequenced in their entirety (fig. 3). The star-shaped phylogeny indicates a roughly equivalent divergence of each strain from all others, excepting the cases where the viruses are closely related. The four Australian isolates fail to form a monophyletic group, indicating that the subtype B epidemic in that country has been the product of the multiple introduction of diverse viral strains, rather than having spread from a small number of index infections.

Discussion

The HIV-B primers were designed to highly conserved regions of the HIV-1 genome and have been applied suc- cessfully to over ten subtype B isolates (table 1). Roughly half of the primers have also been used with non-B sub- types (E, G and A). Both cultured material and patient PBMC have been successfully used as the primary tem- plate for long-range PCR (fig. 2). The advantages of this technique over previously described methods are several. The optional removal of viral culture and cloning of the long-range product saves several weeks' work, as well as avoiding selective effects on the viral sequence from both these procedures. The use of M 13-tailed primers for both amplification and sequencing saves further time and allows high-quality sequence to be generated from both DNA strands. The overlap of several hundred base pairs between the amplimers enabled rapid contig assembly and confirmation that sequences have been derived from the same genomic template.

132 J Biomed Sci 2000;7:128-135 Oelrichs/Lawson/Coates/Chatfield/Deacon/ McPhee

MBC200 NL4.3 MBC925 AD8

V2

iC~NIiTTiRIRi:iNK~Q WPIDNDR~NTSYRLiV$~NTSVITQ ACPKVSFEPI ACPKVSFEPI

!y!K~PV!DK I~:~NTSYKLi !i$~TSVITQ ACPKVSFEPI I~SIFNI~T<~:~;i~I:~ID!mklfi~ALF:II<k~L~WPI!D,N~]iD~42NTSYRLIJ!~N~NTSTITQ ACPKVSFEPI

221

212 220 210

MBC200 NL4.3 MBC925 ADS

MBC200 NL4.3 MBC925 AD8

PIHYCAPAGF AILKCRDKKF NGTGPCKGVS TVQCTHGIRP VVSTQLLLNG SLAEEEVVIR PIHYCAPAGF AILKCNNKTF NGTGPCTNVS TVQCTHGIRP VVSTQLLLNG SLAEEDVVIR PIYYCAPAGF AILKCKDEKF NGKGLCTNVS TVQCTHGIRP VVSTQLLLNG SLAEGEVIIR PIHYCTPAGF AILKCKDKKF NGTGPCKNVS TVQCTHGIRP VVSTQLLLNG SLAEEEVVIR

SENFTNNAKT IIVQLNEAIA SANFTDNAKT IIVQLNTSVE SENITNNAKT IIVQLKDPVE SSNFTDNAKN IIVQLKESVE

306 %/3 320

I~CTRPSNST GQS IRI'~GQ RRAFYAYGKI I I~CTR~NNNT

I I~CTRPN~NT SKS IHi ~iGP GRAF~TTGDi

281 272 280 270

338 330 337 327

Fig. 4. Alignment of the V2-V3 region of the predicted Env proteins. Variable regions are boxed between flanking cysteine residues. The V2 insertion of MBC200 and the charged residues at NL4.3 positions 306 and 320 are indi- cated. The GPG triplet central to the proposed CCR5 binding consensus motif is highlighted.

The amino acid sequences deduced from these data present a number of features that correlate with the observed in vitro phenotypes (fig. 4). HIV-1MBC200 exhib- its an extended V2 region, containing several potential N- linked glycosylation sites [ 12, 17] and positively charged amino acids [7] as well as positively charged amino acids in the critical V3 residues 306 and K320 [8], consistent with an SI phenotype. By contrast, HIV-1MBC925 has V3 residues H306 and the negatively charged D320, consis- tent with NSI phenotype. Both isolates have conserved residues shown to be important in binding the chemokine receptor CCR5, as deduced from mutagenesis and mod- eling of the crystal structure [40, 50]; however, the V3 consensus CCR5 binding sequence deduced by Xiao et al. [53] is conserved only in HIV-1MBC925, despite the apparent use of this receptor by both isolates in HOS.CD4.CCR5 cells.

The N-terminal region of Nefhas been shown to play a role in CD4 down-modulation [3, 23] and it is worth not- ing the predicted 9-amino acid insertion that has arisen by duplication in this region for HIV-1MBC200. However, it is not yet possible to assign a clear functional significance to this or the many other scattered mutations and differ- ences that occur across the genomes.

Thus, collectively from the tissue of origin, cyto- pathicity, co-receptor usage and CD4+ T-cell killing, HIV-1MBC925 clearly represents an M-tropic/CCR5-using

strain and HIV-1MBC200 a T-tropic/CXCR4-using strain. Molecular data confirmed and extended these phenotyp- ic data. A notable characteristic is the NSI phenotype but rapid/high replication kinetics for HIV-1MBC925, ex- panding those typically observed for NSI/M tropic viruses [2, 1 1]. CD4+ T-cell killing seen with HIV-1Nm.3 and HIV-tMBC200 also represents an excellent marker for viral tropism, extending previous data [38, 54].

Phylogenetic analysis performed on full-length se- quences permits a fine examination of the relationships between viral strains, that is more sensitive than those based on smaller, subgenomic fragments. In this case, as expected, full-length isolates derived from the same pa- tient cluster with maximum certainty (ACH1 and 2; JRCSF and JRFL; YU2 and YU l 0) as do the five related sequences HXB2, LAI, LW12.3, PV22 and TH475 (fig. 3). The relationship between isolates NY5 and CAM 1 is surprising as no apparent link exists in the deri- vation of these strains. The Australian isolates reflect the diversity of the epidemic by their separation in neighbor- joining analysis.

Studies in HIV-1 molecular epidemiology and viral evolution will increasingly require detailed sequence in- formation at the level of the entire genome. The study presents a technique whereby such information may be rapidly obtained and describes its application to two Aus- tralian HIV-1 strains. These data expand the database of

Rapid Full-Length Analysis of Australian HIV-1 Isolates

J Biomed Sci 2000;7:128-135 133

fully sequenced primary clinical isolates. It is likely that the application of these techniques to the broader epi- demic will reveal previously undetected patterns of HIV- 1 transmission and evolution.

HIV-1 sequences have been deposited in the Genbank database, accession numbers HIV-1MBC200: AF042100, HIV- 1MBC925:AF042101.

Acknowledgments

This work was supported in part by an Australian government grant to the National Centre in HIV Virology Research and the Mac- farlane Burner Centre for Medical Research Fund. The authors thank Dr. Nelson Michael for advice concerning Iong-range PCR and Dr. Sally Land for virus isolation.

References

I Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA. CC CKR5: a REANTES, MIP-lct, MIP-I~ recep- tor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1958;I996.

2 Asj6 B, Morfedt-MS.nson L, Albert J, Biberfeld G, Karlsson A, Lidman K, Feny5 EM. Replica- tive capacity of human immunodeficiency vi- rus from patients with varying severity of HIV infection. Lancet ii:660-662; 1986.

3 Chen BK, Gandhi RT, Baltimore D. CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef J Viro170:6044-6053;1996.

4 Cheng-Mayer C, Lin R, Landau NR, Stamata- tos L. Macrophage tropism of human immu- nodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor. J Virol 71:1657-1661; 1997.

5 Cho MVW, Lee MK, Carney MC, Berson JF, Doms RW, Martin MA. Identification of deter- minants on a dualtropic human immunodefi- ciency virus type I envelope glycoproteins that confer usage of CXCR4. J Viro172;2509-2515; 1998.

6 Collman RJ, BalIiet W, Gregory SA, Friedman H, Kolson DL, Nathanson N, Srinivasan A. An infectious molecular clone of an unusual mac- rophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol 66:7517-7521; 1992.

7 Cornelissen M, Hogervorst E, Zorgdrager F, Hartman S, Goudsmit J. Maintenance of syn- cytium-induced phenotype of HIV type 1 is associated with positively charged residues in the HIV type 1 gp 120 V2 domain without fixed positions, elongation, or relocated N-linked glycosylation sites. AIDS Res Hum Retrovi- ruses 11:1169-1175;1995.

8 De Jong J J, De Ronde A, Keulen W, Tersmette M, Goudsmit J. Minimal requirements for the human immunodeficiency virus type 1 V3 do- main to support the syncytium-inducing phe- notype: Analysis by single amino acid substitu- tion. J Viro166:6777-6780;1992.

9 Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sut- ton RE, Hill CM, Davis CB, Peiper SC, SchalI TJ, Littman DR, Landau NR. Identification of a major co-receptor for primary isolates of HIV- 1. Nature 381:661-666; 1996.

10 Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Mad- don P J, Koup RA, Moore JP, Paxton WA. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381: 66%673; 1996.

11 Feny5 EM, Morfeldt-Mfinson L, Chiodi F, Lind B, yon Gegerfelt A, Albert J, Olausson E, Asj6 B. Distinct replicative and cytopathic characteristics of human immunodeficiency vi- res isolates. J Viro162:4414-4419; 1988.

12 Fouchier RA, Groenink M, Kootstra NA, Ters- rnette M, Huisman HG, Miedema F, Schuite- maker H. Phenotype-associated sequence vari- ation in the third variable domain of the hu- man immunodeficiency virus type 1 gpl20 molecule. J Viro166:3183-3187; 1992.

13 Gao F, Robertson DL, Carruthers CD, Morri- son SG, Jian B, Chen Y, Barre-Sinoussi F, Girard M, Srinivasan A, Abimiku AG, Sharp PM, Hahn BH. A comprehensive panel of near- full-length clones and reference sequences for non-subtype B isolates of human immunodefi- ciency virus type l. J Virol 72:5680-5698; 1998.

14 Ghosh SK, Fultz PN, Keddie E, Saag MS, Sharp PM, Hahn BH, Shaw GM. A molecular clone of HIV-1 tropic and cytopathic for hu- man and chimpanzee Iymphocytes. Virology I94:858-864;1993.

15 Graf M, Shao Y, Zhao Q, SeidI T, Kestler J, Wolf H, Wagner R. Cloning and characteriza- tion of a virtually full-length HIV-1 genome from a subtype B'-Thai strain representing the most prevalent B-clade isolate in China. AIDS Res Hum Retroviruses 14:285-288;1998.

16 Greenway AL, McPhee DA, Grgacic E, Hewish D, Lacantoni A, Macreadie I, Azad AA. Nef27 but not the Nef 25 isoform of human immu- nodeficiency virus type 1 pNIA.3 isolate down- regulates surface CD4 and IL-2R expression in peripheral blood mononuclear cells and trans- formed T-cells. Virology' 198:245-256; 1994.

17 Groenink M, Fouchier RA, Broersen S, Baker CH, Koot M, van't Wout AB, Huisman HG, Miedema F, Tersmette M, Schuitemaker H. Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science 260:1513- 1516;1993.

18 GuiUon C, Bedin F, Fouchier RA, Schuitemak- er H, Gruters RA. Completion of nucleotide sequence ofnon-syncytium-induced and syncy- tiara-inducing tlIV type 1 variants isolated from the same patient. AIDS Res Hum Retro- viruses 11:1537-1541;1995.

19 Gurgo C, Guo HG, Franchini G, Aldovini A, Collalti E, Farrell K, Wong-StaaI F, Gallo RC, Reitz MS. Envelope sequences of two new United States HIV-1 isolates. Virology 164: 531-536;1988.

20 Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 36:365-369;1967.

21 HIV sequence database at the Los Alamos Na- tional Laboratory (http://hiv-web.lanI.gov/). These figures are based on data available 30/ 6/99.

22 Huet T, Dazza MC, Brun-Vezinet F, Roelants GE, Wain-Hobson S. A highly defective HIV-1 strain isolated from a healthy Gabonese indi- vidual presenting an atypical Western blot. AIDS 3:707-715;1989.

23 Iafrate AJ, Bronson S, Skowronski J. Separable functions of Nef disrupt two aspects of T cell receptor machinery: CD4 expression and CD3 signaling. EMBO J 16:673-684; 1997.

24 Kemp DJ, Churchill M J, Smith DB, Biggs BA, Foote SJ, Peterson MG, Samaras N, Deacon NJ, Doherty R. Simplified colorimetric analy- sis of polymerase chain reactions: Detection of HIV sequences in AIDS patients. Gene 94: 223-228; 1990.

25 Kiernan R, Marshall J, Bowers R, Doherty R, McPhee D. Kinetics of HIV-I replication and intracellular accumulation of particles in HTLV-I-transformed cells. AIDS Res Hum Retroviruses 6:743-752;1990.

26 Koyanagi Y, Miles S, Mitsuyasu RT, Merrill JE, Vinters HV, Chen IS. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236:819- 822; 1987.

27 Laukkanen T, Janssen W, Carr J, Albert J, Leinikki PO, McCutchan FE, Salminen MO. Inter-subtype recombinant strains of HIV-1 are frequently found among new full-length se- quences. In: Conference Record of the t2th World AIDS Conference, Geneva 1998; abstr 1 l170, p 16.

134 J Biomed Sci 2000;7:128-135 Oelrichs/Lawson/Coates/Chatfield/Deacon/ McPhee

28 Lee TH, Sunzeri FJ, Tobler LH, Williams BG, Busch MP. Quantitative assessment of HIV-I DNA load by coamplification ofNIV-1 gag and HLA-DQ-a genes. AIDS 5:683-691; 1990.

29 Li Y, Kappes JC, Conway JA, Price RW, Shaw GW, Hahn BH. Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: Identification of replication-competent and -defective viral genomes. J Virol 65:3973- 3985;1991.

30 McPhee D, Greenway A, Holloway G, Smith K, Deacon N, Pemberton L, Brew B. Anoma- lies in Nef expression within the central ner- vous system of HIV-l-positive individuals/ AIDS patients with or without AIDS dementia complex. J Neurovirol 4:291-300; 1998.

31 Muesing MA, Smith DH, Cabradilla CD, Ben- ton CV, Lasky LA, Capon DJ. Nucleic acid structure and expression of the human AIDS/ lymphadenopathy retrovirus. Nature 313:450- 458;t985.

32 Neate EV, Pringle RC, Jowett JBV, Healey DS, Gust ID. Isolation of HIV t?om Australian patients with AIDS, AIDS-related conditions and healthy antibody-positive individuals. Aust NZ J Med 17:461-466;1987.

33 Neumann M, Felber BK, Kleinschmidt A, Froese B, Erfle V, Pavlakis GN, Brack-Werner R. Restriction of human immunodeficiency vi- rus type t production in a human astrocytoma cell line is associated with a cellular block in Rev function. J Viro169:2159-2167;1995.

34 Oelrichs R, Tsykin A, Rhodes D, Solomon A, Ellett A, McPhee D, Deacon N. Genomic se- quence of HIV-1 from four members of the Sydney Blood Bank cohort of long-term non- progressors. AIDS Res Hum Retroviruses 14: 811-814;1998.

35 Oelrichs R, Workman C, Laukkanen T, McCutchan F, Deacon N. A novel subtype A/ G/J recombinant fulMength HIV-I genome from Burkina Faso. AIDS Res Hum Retrovi- ruses 14:1495-1500;1998.

36 Oelrichs R, Juarez J, Alali M, Cunningham A, Stewart G, Naif H. Unpubl data, 1999.

37 Ou CY, Kwok S, Mitchell SW, Mack DH, Sninsky J J, Krebs JW, Feorino P, Warfield D, Schochetman G. DNA amplification for direct detection of HIV- 1 in DNA of peripheral blood mononuclear cells. Science 239:295-297; 1988.

38 Pal A, Greenough TC, Sullivan JL, Somasun- damn MJ. In vitro characterization of adult primary human immunodeficiency virus type 1: Demonstration of distinctive single-cell kill- ing phenotypes in spite of similar levels of viral replication. J Infect Dis 176:933-940;1997.

39 Reitz M, Hall L, Robert-Guroff M, Lauten- berger J, Hahn B, Shaw G, Kong L, Weiss S, Waters D, Gallo R, Blattner W. Viral variabili- ty and serum antibody response in a laboratory worker infected with HIV type 1 (HTLV type IIIB). AIDS Res Hum Retroviruses 10:1143- 1155; 1994.

40 Rizzuto CD, Wyatt R, Heruandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, Sodroski J. A conserved HIV gpl20 glycoprotein struc- ture involved in chemokine receptor binding. Science 280:1949-1953; 1998.

41 Robertson D, Gao F, Hahn B, Sharp P. Inter- subtype recombinant HIV-1 sequences, p III: 25-30. In: Korber B, Hahn B, Foley B, Mellors J, Leitner T, Myers G, McCutchan F, Kuiken C, eds. The Human Retroviruses and AIDS 1997 Compendium. Los Alamos National Lab- oratory, 1997.

42 Salminen MO, Koch C, Sanders-Buell E, Eh- renberg PK, Michael NL, Carr JK, Burke DS, McCutchan FE. Recovery of virtually full- length HIV- 1 provirus of diverse subtypes from primary virus cultures using the polymerase chain reaction. Virology 213:80-86; 1995.

43 Sanchez-Pescador R, Power MD, Barr PJ, Steimer KS, Stempien MM, Brown-Shimer SL, Gee WW, Renard A, Randolph A, Levy JA, Dina D, Luciw PA. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227:484-492; 1985.

44 Sauermann U, Schneider J, Mous J, Brunck- horst U, Schedel I, Jentsch KD, Hunsmann G. Molecular cloning and characterization of a German HIV-1 isolate. AIDS Res Hum Retro- viruses 6:813-823; 1990.

45 Shibata R, Hoggan MD, Broscius C, Englund G, Theodore TS, Buckler-White A, Arthur LO, Israel Z, Schultz A, Lane HC, Martin MA. Iso- lation and characterization of a syncytium- inducing, macrophage/T-cell line-tropic hu- man immunodeficiency virus type 1 isolate that readily infects chimpanzee cells in vitro and in vivo. J Viro169:4453-4462; 1995.

46 Siebenkotten G, Leyendeckers H, Christine R, Radbruch A. Isolation of plasmid DNA from mammalian cells with QIAprep. Qiagen News 2/95:11-12;1995.

47 Starcich BR, Hahn BH, Shaw GM, McNeely PD, Modrow S, Wolf H, Parks ES, Parks WP, Josephs SF, Gallo RC, Wong-Staal F. Identifi- cation and characterization of conserved and variable regions in the envelope gene of HTLV- III/LAV, the retroviruses of AIDS. Cell 45: 637-648; 1986.

48 Theodore TS, Englund G, Buckler-White A, Buckler CE, Martin MA, Peden KW. Construc- tion and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions. AIDS Res Hum Retroviruses 10:191-194;1996.

49 Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17; 1985.

50 Wang WK, Dudek T, Zhao YJ, Brumblay HG, Essex M, Lee TH. CCR5 coreceptor utilization involves a highly conserved arginine residue of HIV type 1 gp 120. Proc Natl Acad Sci USA 95: 5740-5745;1998.

51 Willey RL, Rutledge RA, Dias S, Folks T, The- odore T, Buckler CE, Martin MA. Identifica- tion of conserved and divergent domains with- in the envelope gene of the acquired immuno- deficiency syndrome retrovirus. Proc Nati Acad Sci USA 83:5038-5042;1986.

52 Wong-Staal F, Gallo RC, Chang NT, Ghrayeb J, Papas TS, Lautenberger JA, Pearson ML, Petteway SR, Ivanoff L, Baumeister K, White- horn EA, Rafalski JA, Dorm~ ER, Josephs S J, Starcich B, Livak KJ, Patarca R, Haseltine WA, Ratner L. Complete nucleotide sequence of the AIDS virus. HTLV-III. Nature 313:277- 284;1985.

53 Xiao L, Owen SM, Goldman I, Lal AA, deJong J J, Goudsmit J, Lal RB. CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically dis- tinct global HIV-1 isolates: Delineation of con- sensus motif in the V3 domain that predicts CCR-5 usage. Virology 240:83-92;1998.

54 Yu X, McLane MF, Ratner L, O'Brien W, Coll- man R, Essex M, Lee TH. Killing of primary CD4+ T cells by non-syncytium-inducing mac- rophage-tropic human immunodeficiency vi- rus type 1. Proc Natl Acad Sci USA 91:10237- 10241;1994.

55 Zhu T, tto DD. Was HIV present in 19597 Nature 374:503-504; 1995.

Rapid Full-Length Analysis of Australian HIV-1 Isolates

J Biomed Sci 2000;7:128-135 135