Open Access

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

  • Marieke Pingen1, 2,
  • Annemarie MJ Wensing2,
  • Katrien Fransen3,
  • Annelies De Bel4,
  • Dorien de Jong2,
  • Andy IM Hoepelman5,
  • Emmanouil Magiorkinis6,
  • Dimitrios Paraskevis6,
  • Maja M Lunar7,
  • Mario Poljak7,
  • Monique Nijhuis2 and
  • Charles AB Boucher1Email author
Retrovirology201411:105

https://doi.org/10.1186/s12977-014-0105-9

Received: 25 March 2014

Accepted: 5 November 2014

Published: 29 November 2014

Abstract

Background

In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity.

Results

We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).

To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses.

Conclusions

We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Keywords

HIVDrug resistanceTransmissionEvolutionReversionPersistenceCompensatory fixation

Background

The viral enzymes reverse transcriptase (RT) and protease were the first targets of antiretroviral therapy and the most commonly used drug regimens still aim at inhibiting these viral proteins [1]. In resource-rich settings, drug resistance mutations in protease and RT are detected in 10-15% of newly diagnosed HIV patients [2],[3].

The majority of transmitted drug-resistant viruses contain limited resistance profiles to single drug classes. Nucleoside RT inhibitor (NRTI) mutations are the most frequently observed transmitted drug resistance mutations (TDRM). Especially thymidine analogue mutations (TAMs) M41L and T215 variants, that have been selected by drugs extensively used in the past, are often observed in newly diagnosed patients [4]. A worrying trend is the increased prevalence of non-nucleoside RT inhibitor (NNRTI) related mutations in newly diagnosed patients [3],[5], as single NNRTI mutations, such as the frequently observed K103N mutation, can result in high levels of resistance against first generation NNRTIs [6]. In protease, M46I/L and L90M are the most frequently observed TDRM [2],[3]. When present in combination with other protease drug resistance mutations, both M46I/L and L90M are related to reduced susceptibility to several protease inhibitors (PIs) [6].

It is generally acknowledged that most drug resistance mutations decrease the replicative capacity (RC) of HIV-1 [7],[8]. As such, in the absence of drugs TDRM can revert to wild type, thereby increasing viral RC. Indeed, follow-up of untreated individuals diagnosed with a drug resistant HIV variant revealed that certain mutations with a detrimental effect on the viral RC, such as M184V in RT, after transmission to a new host often revert rapidly in the plasma [9],[10]. In addition, the use of very sensitive assays shows that minority drug resistance mutations are frequently found in untreated individuals, suggestive of reversion after transmission [11],[12].

However, follow-up of patients diagnosed with HIV-1 harboring TDRM has revealed that TAMs, PI- and NNRTI-related TDRM often persist for prolonged periods [10],[13]-[25]. The mechanisms explaining persistence have not been fully resolved. Based on the available literature [13],[15]-[25], we have previously proposed two possible mechanisms to explain persistence of TDRM [9]. When the effect of the TDRM on the RC is very small, reversion may take a very long time. Alternatively, when the TDRM decreases the RC considerably the presence or selection of additional compensatory mutations can prevent reversion of the TDRM.

The aim of our study was to gain more insight in the mechanisms causing persistence of drug resistant HIV-1 variants after transmission. Therefore, we investigated the molecular evolution of HIV-1 protease and RT harboring the most frequently observed TDRM in great detail. The majority of TDRM persisted during the follow-up, and only few additional polymorphisms were selected during this period. Most patient-derived viruses had a higher RC than the corresponding site-directed mutant viruses, indicating that persistence can be explained by a high replication capacity of most transmitted drug resistant HIV-1 variants.

Results

Patients diagnosed with a transmitted drug resistant HIV-1 variant

To investigate the in vivo evolution of transmitted drug resistant HIV variants, we selected 31 patients from four European countries (Belgium, Greece, the Netherlands, Slovenia) who were diagnosed in 2001 to 2008 with an HIV variant harboring a frequently observed TDRM (prevalence >5% in patients diagnosed with HIV-1 harboring TDRM in the SPREAD-programme). Patients were included if a plasma sample was available at one year (10–14 months) after diagnosis if therapy was not yet initiated. If available, a third time point before start of treatment was investigated. Prior negative HIV tests were available for 14 patients, revealing that at least nine patients had been infected for less than two years. The majority of the patients were men having sex with men (MSM), which is the most important route of transmission in Western Europe. The median plasma HIV-RNA in our group of patients was 4.6 log copies/ml, comparable to the median HIV-RNA observed in the SPREAD-programme in 2002–2006 (4.8 log copies/ml). The median baseline CD4 count was 653 cells/mm3, which is higher than the median observed in the SPREAD programme (343 cells/mm3) [3].

Surveillance studies demonstrated that most transmitted drug resistant HIV-1 variants harbor resistance against a single drug class [3],[4]. In line with this observation, only 3/31 of the patients selected for this study had been diagnosed with an HIV-1 variant resistant to multiple drug classes. A total of 55 mutations at positions included in the WHO list for surveillance of transmitted drug resistant HIV-1 [26] were observed in the transmitted viruses at baseline. A single TDRM was detected in 10/16 patients with viruses harboring only NRTI-related TDRM, for the other six patients a profile of two to four TDRM was observed. The vast majority of NRTI-related TDRM were TAM-related mutations. In six of the selected patients viral variants containing a single NNRTI-related TDRM were observed. Six patients were diagnosed with HIV-1 harboring a single PI-related TDRM (Table 1). In addition to TDRM, polymorphisms were present in all baseline sequences. For variants containing RT TDRM, the median number of RT polymorphisms was 7 (range: 4–21) when compared to HXB2 and 6 to consensus B (range: 2–19). Viruses harboring PR resistance mutations had a median of 6 baseline polymorphisms in protease when compared to HXB2 (range: 4–9) and median of 5 when compared to consensus B (range: 3–8).
Table 1

Patient characteristics, resistance mutations and evolution

ID

Gender

Last negative HIV test

Country of origin

Diagnosis

Risk group

Months after first analysis

Plasma HIV RNA

(copies/ml)

CD4 count

Sub-type

Resistance Profile PR

Resistance Profile RT

p-distance

p-value dN/dS

Transmitted variants harboring only NRTI-related mutations

P01

Male

 

NL

May 2007

MSM

0

>750000

461

B

 

M41L

  
      

10

    

M41L

0.001

1.000

      

16

    

M41L

0.002

0.290

P02

Male

 

NL

Jan 2008

MSM

0

21800

423

B

 

M41L

  
      

12

    

M41L

0.005

0.225

      

28

    

M41L

0.005

0.225

P03

Male

Jan 2004

BE

Jun 2005

MSM

0

41000

483

B

 

M41L

  
      

11

    

M41L

0.000

1.000

      

32

    

M41L

0.002

0.152

P04

Male

 

NL

Feb 2007

MSM

0

102000

322

B

 

L210LS

  
      

11

    

-

0.000

1.000

      

25

    

-

0.002

1.000

P05

Male

 

SL

Jun 2001

MSM

0

12267

950

B

 

T215D

  
      

12

    

T215D

0.011

0.060

      

99

    

T215D

0.007

0.428

P06

Male

Mar 2005

SL

Feb 2006

MSM

0

797000

953

B

 

T215S

  
      

14

    

T215S

0.000

1.000

      

21

    

T215S

0.000

1.000

P07

Male

 

NL

Sep 2008

MSM

0

36300

521

B

 

T215D

  
      

11

    

T215D

0.000

1.000

      

27

    

T215D

0.000

1.000

P08

Male

Sep 2004

NL

Dec 2004

MSM

0

583000

596

B

 

T215IT

  
      

13

    

-

0.000

1.000

P09

Male

Sep 2006

NL

Sep 2007

MSM

0

158000

678

B

 

T215AT

  
      

13

    

T215AT

0.001

0.294

      

20

    

T215A

0.000

1.000

P10

Male

Oct 2003

NL

Jan 2005

MSM

0

89800

289

B

 

K219N

  
      

11

    

K219N

0.000

1.000

      

44

    

K219N

0.000

1.000

P11

Male

 

BE

Mar 2006

HSX

0

318000

966

B

 

D67N T215C

  
      

13

    

D67N T215C

0.001

0.291

P12

Male

 

NL

Feb 2007

MSM

0

55900

609

B

 

D67G T215C K219E

  
      

11

    

D67G T215C K219E

0.007

0.156

      

24

    

D67G T215C K219E

0.000

1.000

P13

Male

Jul 2004

NL

Nov 2007

MSM

0

294000

531

B

 

D67G T215C K219E

  
      

12

    

D67G T215C K219E

0.000

1.000

      

14

    

D67G T215C K219E

0.000

1.000

P14

Male

Apr 2005

NL

Jun 2005

HSX

0

750000

577

B

 

D67G T215C K219E

  
      

14

    

D67G T215C K219E

0.000

1.000

P15

Male

 

NL

Aug 2005

MSM

0

81000

470

B

 

M41L T69S T210E T215ST

 

1.000

      

11

    

M41L T69S T210DE T215ST

0.000

1.000

      

39

    

M41L T69S T210E T215ST

0.000

1.000

      

77

    

M41L T69S T210E T215ST

0.000

1.000

P16

Male

Mar 2005

NL

Jun 2006

MSM

0

34600

1129

B

 

M41L T69S T210E T215ST

  
      

13

    

M41L T69AS T210E T215ST

0.000

1.000

      

33

    

M41L T69S T210E T215ST

0.001

1.000

      

49

    

M41L T69S T210E T215ST

0.001

1.000

Transmitted variants harboring only NNRTI-related mutations

P17

Male

Feb 2005

NL

Sep 2006

MSM

0

5990

790

B

 

K103N

  
      

12

    

K103N

0.000

1.000

      

30

    

K103N

0.000

1.000

P18

Male

Jun 2004

BE

Apr 2006

MSM

0

39900

648

B

 

K103N

  
      

12

    

K103N

0.000

1.000

      

28

    

K103N

0.000

1.000

P19

Male

 

NL

Sep 2005

MSM

0

21400

359

B

 

K103Q

  
      

12

    

K103Q

0.000

1.000

      

59

    

K103Q

0.001

0.304

P20

Male

1995

SL

Sep 2005

MSM

0

29300

421

B

 

Y181C

  
      

11

    

Y181C

0.000

1.000

      

49

    

Y181C

0.002

0.305

P21

Female

 

GR

Sep 2004

HSX

0

905

699

B

 

G190A

  
      

10

  

B

 

G190A

0.005

0.866

P22

Male

 

GR

Jun 2004

?

0

10500

918

B

 

G190A

  
      

13

  

B

 

G190A

0.005

0.387

Transmitted variants harboring only PI-related mutations

P23

Male

 

NL

Apr 2007

HSX

0

700000

664

B

M46L

   
      

14

   

M46L

 

0.000

1.000

      

22

   

M46L

 

0.000

1.000

P24

Male

Jan 2006

NL

Apr 2008

MSM

0

5170

742

B

M46L

   
      

10

   

M46L

 

0.001

0.310

      

29

   

M46L

 

0.004

0.471

P25

Male

Jul 2005

NL

Aug 2008

MSM

0

421000

409

B

M46L

   
      

14

   

M46L

 

0.000

1.000

      

23

   

M46L

 

0.000

1.000

P26

male

 

NL

Aug 2008

MSM

0

111000

657

B

M46L

   
      

14

   

M46L

 

0.000

1.000

      

26

   

M46L

 

0.001

0.299

P27

male

05-11-04

NL

Apr 2007

MSM

0

18100

699

B

M46L

   
      

13

   

M46L

 

0.000

1.000

      

38

   

M46L

 

0.001

0.306

P28

male

15-02-03

NL

Mar 2005

MSM

0

69000

480

B

L90M

   
      

13

   

L90M

 

0.000

1.000

Transmitted variants harboring mutations against two drug classes

P29

male

 

NL

Dec 2001

MSM

0

288

1468

B

 

D67G Y181CY T215C K219E

  
      

10

    

D67G T215C K219E

0.006

0.148

      

46

    

D67G T215C K219E

0.000

1.000

P30

male

 

NL

Jan 2005

HSX

0

26600

667

B

G73S L90M

K103N

  
      

12

   

L90M

K103N

0.001

0.306

      

18

   

L90M

K103N

0.001

0.306

P31

female

 

GR

Jul 2004

HSX

0

696

1288

B

I54V V82A L90M

M41L D67N L210W T215D

  
      

10

   

F53FL I54V V82A L90M

M41L D67N L210W T215D

0.001

0.293

Abbreviations: PR protease, RT reverse transcriptase, NRTI nucleoside reverse transcriptase inhibitor, NNRTI non- nucleoside reverse transcriptase inhibitor, PI protease inhibitor, BE Belgium, GR Greece, NL the Netherlands, SL Slovenia, HSX heterosexual, MSM Men having sex with men? unknown route of transmission.

In vivoevolution of transmitted drug resistant HIV-1 variants

The vast majority (51/55) of TDRM persisted during the first year of follow-up. For 24/31 patients a third and sometimes a fourth genotypic analysis was performed at a median of 28 months (range: 14–99 months) after the first sample. During this more extensive follow-up period of up to eight years, all resistance mutations present at one year after diagnosis persisted in the plasma (Table 1).

To gain more understanding of in vivo persistence of TDRM, we performed a comprehensive analysis of in vivo viral evolution during the follow-up. Viruses harboring protease drug-resistance mutations selected a median of 1 (range: 0–1) additional polymorphisms in protease during the first year of follow-up. Likewise, viruses harboring drug-resistance mutations in RT selected a median of 1 (range 0–3) additional RT polymorphisms (Table 2). As a measure of evolution at the nucleotide level, the p-distance between baseline and follow-up sequences was calculated. For the majority of patients, this revealed a very low p-distance between baseline and one year, confirming limited viral evolution. In line with this observation, the dN/dS ratio of the viral populations, which is an indicator of selection, did not change significantly in any patient (Table 2). However, in all transmitted viruses at least one change at a polymorphic site was observed, which is described in Table 2.
Table 2

Evolution of transmitted drug resistant HIV variants

ID

Months after first sample

Protease amino acid 4-99

Reverse transcriptase amino acid 41-230

Baseline

Reversion

Additional mutations

Baseline

Reversion

Additional mutations

Transmitted variants harboring only NRTI-related mutations

P01

0

S37N L63P I93L

  

M41L V60I I135T S162C K166R R211G L214F

  
 

10

    

-K166R

+V106IV

 

16

     

V106IV > I

P02

0

T12A K14KR Q18HQ L19IL S37N L63P I93L

  

M41L V60I I135T S162G K166EK I167F R211G L214F

  
 

12

 

-K14KR, −Q18HQ

L19IL > IKLQ

 

-S162G -I167F

K166EK > KR

 

28

 

T12A > AT

L19IKLQ > IL

 

R211G > GR

+T165IT

P03

0

T12A I13V L19I S37NS I64V C67CR

  

M41L V60I F61FS E122K D123E I178L V179IV E203EG Q207EQ L214F

  
 

11

 

-S37NS -I64V > IV -C67CR

+I62IV

 

-F61FS -E203EG

+S162X V179IV > I Q207EQ > KQ + R211KR

 

32

 

T12A > AT

I62IV > V

 

-S162X -I178L -V179I

Q207KQ > EQ

P04

0

E35D S37D D60E I62V L63P A71T I72V I93L

  

K49R V60I V118I E122K D123DE I135R S162D L210LS R211G

  
 

11

  

+I72V > EV

 

-L210LS

D123DE > E S162D > S162X + T200IT + E204EK

 

25

  

+T12AT + K14KR + V77IV

 

-S162X, R211G > GR -E204EK

+T165IT

P05

0

S37N I64V

  

K64R R83K I178L I202V L214F T215D

  
 

12

  

+M36I

 

-K64R

+S68N + E122K

 

99

  

+I13IV + K14KR + K45KR

 

R83K > KR, −I202V

+A158AS + S162T

P06

0

L10I K14EV S37N L63T E65EV I72T V77I I93L

  

E122K I142V D177E Q207E L214F T215S

  
 

14

  

K14EV > E E65EV > V

 

-E122K > EK

 
 

21

      

P07

0

I15L L19V S37N R41K D60E L63P I72IV I93L

  

V60I S68G R83K V90I A98S E122P D123DEG I135L S162C D177E I202IV R211K L214F T215D

  
 

11

  

+M36IM

 

−202IV

D123DEG > DE

 

27

 

-M36IM

L19V > IV

  

D123DE > DEG + T200IT

P08

0

L10I S37N R41K I62V L63S V77I I93L

  

V60I S68G E122K I135V S162NS T165IT Q174HQ G196E R211G L214F T215IT

  
 

13

    

-S162NS -T165IT-T215IT

Q174HQ > H

P09

0

S37N I62V L63T I64L V77I

  

S68T E122K I135V T139A G196E Q197R L214F T215AT

  
 

13

    

-T139A

+H198HR

 

20

  

+R57KR

 

-H198HR

S68T > AT + T139AT + T215AT > A

P10

0

I15V E35D S37D D60E L63P V77I I93L

  

S68K T69N A98S L100LV E122K D123E I135R N136NT Q145E S162C I178M E194D I195L G196E T200A I202V Q207K R211G L214F K219N H221Y K223Q

  
 

11

  

+R41K

 

-L100LV -N136NT

 
 

44

  

+K45KR + R57KR

 

-I135R

+K49KR

P11

0

S37H R41KR R57K Q61D

  

V60I D67N T69E V106I D121Y I135T S162C D177E G196E E203D Q207E R211KR L214F T215C

  
 

13

    

-V106I L214F > FL

+T200IT

P12

0

L10I T12S L19I L63T

  

V60I D67G S68G I135T I178M R211KR L214F T215C K219E

  
 

11

 

-L10I

L19I > T

 

-R211KR

+E122EK

 

24

  

+L10I L19T > I + I62IV

 

-E122EK -I135T

+Q207LQR + R211KR

P13

0

T12S L19I L63T I64IM

  

V60I D67G S68G A158S I178M L214F T215C K219E

  
 

12

  

I64IM > M

  

+E40Q

 

14

    

-E40Q

 

P14

0

T12S L19T L63T

  

V60I D67G S68G I135IT I178M L214F T215C K219E

  
 

14

     

+E122EK I135IT > T

P15

0

L19I E35D S37N R57KR L63P V77IV I93L

  

M41LT69S D86DE E122K S162C I178L E204DE Q207EKQ L210E R211K L214F T215ST

  
 

11

 

-R57KR

V77IV > I

  

Q207EKQ > KQR L210E > DE

 

39

 

V77I > IV

S37N > DN + R57KR

  

+V60VI + I195IL Q207KQR > x L210DE > E R211K > KN

 

77

 

’-R57KR

  

S162C > CS

E204DE > DEKNR211KN > K

P16

0

L19I E35D S37NS L63P V77IV I93L

  

M41L T69S D86E K104KR E122K S162C I178L E204DE Q207KQR L210E R211DEKN L214F T215S

  
 

13

  

I72IM

 

-K104KR

T69S > AS S162C > CW

 

33

 

-I72IM

E35D > DEKN S37NS > N V77IV > I

 

-E204DE

T69AS > S S162CW > W + E194DE Q207KQR > R R211DEKN > D

 

49

  

E35DEKN > DE

 

-E194DE

Q207R > QR R211D > DEKN

P17

1

E35D R41K L63P I93L

  

K103N E122K D123E R211K L214F

  
 

12

     

+K173KT + D177DN

 

30

  

+S37N

 

-K173KT

+Q174HQ + Q207QR R211K > KQ

Transmitted variants harboring only NNRTI-related mutations

P18

0

L10IV I13IV I15IV L19IL I62V L63PS I64LV C67S V77I

  

K64R K103N E122K D123E K173EK Q174QR V179I T200A R211K L214F

  
 

12

 

-L19IL

L10IV > I L63PS > X I64LV > V

 

-K173EK -Q174QR

+D177DN

 

28

     

+R72RS + Q174QR

P19

0

L10I I15V S37T R41K C67G G68E H69R

  

V60I K103Q E122K D123E I142V R211K L214F

  
 

10

      
 

12

      
 

59

  

G68E > D

  

+ T200IT

P20

0

T12N K14R S37N R41KR I64V

  

E122K D123E I135T Y181C T200A I202V R211K L214F

  
 

11

 

S37N > NS

    
 

49

 

K14R > KR -R41KR

+E35D S37NS > N + L63HQ

 

E122K > EK I135T > IT

D123E > AE

P21

0

I13V S37NT L63P A71AG

  

I50N G51W P52A V60IV R83K A98AG K101H S105LS D177E V179I G190A R211K L214F

  
 

10

 

-A71AG

S37NT > NST

 

-I50N -G51Q -P52A -A98AG -S105LS

V60IV > I + E122K K173EK

P22

0

I13V M36T S37N L63P

  

S48Q R83KR K101H D123DE D177E V179I G190A L214F H235R

  
 

13

  

M36T > IMT

 

-S48Q -H235R

D123DE > DEKN + S162CS

P23

0

S37N M46L D60E I62V L63S I72V V77I I93L

  

K49R V60I V118I I135R E169D R211G L214F

  
 

14

     

+F87FL + E204EK

 

22

    

V60I > IV -F87FL

-E204EK

 

P24

0

E35DE S37N M46L D60E I62V L63S I93L

  

K49KR V60I V118I E122K I135R R211G

  
 

10

 

-E35DE -I93L

+K70KR

 

-K49KR

 
 

29

 

-K70KR

   

+K104KR + S162C

P25

0

E35D S37N M46L D60E I62V L63S I93L

  

K49R V60I V118I E122K I135R R211G

  
 

14

  

+R41KR

  

+D123E

 

23

  

L63S > PS I93L > IL

  

D123E > DEKN + I178ILV

Transmitted variants harboring only PI-related mutations

P26

0

E35D S37N M46L D60E I62V L63S I93L

  

K49R V60I V118I E122K D123DN I135R R211G

  
 

14

  

+L19IL

 

-D123DN

+T165IT

 

26

  

L19IL > X + A71AV

  

T165IT > I + E204EK

P27

0

E35D S37N M46L D60E I62V L63S I93L

  

K49R V60I V118I E122K I135R N136NT S162NS I167IT R211G

  
 

13

  

L63S > APS

 

-N136NT -S162NS -I167IT

 
 

38

 

-E35D

L63APS > A

   

P28

0

L19T S37N L63P L90M I93L

  

E122KT200A L214F K220X

  
 

13

 

S37N > NS

  

-K220X

 

Transmitted variants harboring mutations against two drug classes

P29

0

T4IT T12S L19IV L63X

  

V60I D67G S68G K70KR I178M Y181CY L214F T215C K219E

  
 

10

 

-T4IT

+L10I L19IV > I L63X > T

 

-K70KR -Y181CY

+I135IT + E204EG + R211KR

 

46

 

-L10I

T12S > PS + G16AG L19I > IV + M36IM L63T > PT + I64IV

 

-E204EG -R211KR

I135IT > T

P30

0

L10I I13V I15V I62V L63P G73S L90M

  

V60I A98S K103N D121Y D123E I135T R172KR L214F

  
 

12

 

-G73S

  

I135T > IT -R172KR

 
 

18

  

+S37NS

  

+K102KR

P31

0

L10I I15V K20R E35D M36I S37N I54V Q58E L63P A71V V82A L90M

  

M41L K43N V60I D67N E122P I135T E138A I142V L210W R211M L214F T215D

  
 

10

  

+F53FL

 

-L214F

+T139I + I178IV

Patient-derived sequences are compared to HXB2. Bold positions indicate positions related to drug resistance, italics indicate polymorphisms of HXB2 compared to consensus B.

Impact of frequently observed TDRMs on in vitroRC

We determined the impact of TDRM on viral RC by introducing frequently observed drug-resistance mutations M46I, M46L or L90M in protease or M41L, M41L + T215Y or K103N in RT in the background of the lab strain HXB2 by site-directed mutagenesis (Figure 1). Viruses were named according to mutations and origin; the prefix “SDM” indicates site-directed mutagenesis. The RC of all viral variants was determined in primary peripheral blood mononuclear cells (PBMCs), which are natural target cells for HIV. Site-directed mutants HIV-M184V, −I and –T with a known impact on RC were used as controls, and to enable comparison of RC between various experiments [27]. The difference in RC between HIV-WT, −M184V and -M184I has been demonstrated to be biologically relevant in vivo [28],[29].
Figure 1

Impact of frequently observed transmitted drug-resistance mutations on viral replicative capacity. The replicative capacity of site-directed mutant (SDM) viruses and patient-derived viruses was determined by infecting donor peripheral blood mononuclear cells with equal amounts of viral p24. In all experiments, control viruses HIV-M184V, −M184I and –M184T and wild type (WT) HIV were used as reference viruses. Representative experiments are shown in A-C and D-F. Error bars indicate standard deviation (SD) of mean within one experiment. Four biological replicates were performed for all viruses. (A-C) Replicative capacity of SDM-viruses (B, C) compared to control viruses (A). (D-F) Replicative capacity of patient-derived viruses (E, F) compared to control viruses (D). RC of WT and control viruses (A, D) is indicated in the corresponding graphs by a square, and the range in RC of WT and M184T by the grey area. (G) The median p24 production of both experiments as a percentage of WT in the corresponding experiment for all protease or reverse transcriptase mutant viruses. Error bars indicate range (n = 4).

All mutations caused a decrease in RC as compared to HIV-WT, except for mutation L90M in protease. The reduction in RC of the M41L, M41L + T215Y and K103N variants was comparable to each other, and to controls HIV-M184V and -I. M46I and M46L in protease resulted in the most severe reduction of RC (Figure 1).

In vitroRC of patient-derived HIV-1 variants harboring frequently observed TDRM

Subsequently, the RC of frequently observed TDRM was determined in their natural genetic background (Figure 1). We constructed recombinant viruses using patient-derived protease containing M46L, M46I or L90M, or patient-derived n-terminus of RT containing M41L or K103N into HXB2. In addition, two more complex transmitted viruses were studied: a protease-variant containing I54V + V82A + L90M and an RT-variant carrying M41L + T69S + L210E + T215S. Patient-derived clones are indicated by the prefix “p”, followed by the TDRM.

The RC of p46I and p46L was similar to controls HIV-M184I and –V, indicating a diminished replication. The RT variant pK103N had an RC comparable to HIV-WT and the RC of pL90M was higher than HIV-WT. For M41L, it has been described that V60I and S162A function as compensatory mutations in transmitted HIV-1 variants [30]. We selected a patient-virus with M41L but without the potential compensatory mutations (pM41L). In this genetic background, the viral RC was as low as HIV-M184T and even lower than SDM-M41L. However, in vivo the variant containing this M41L mutation persisted for 8 months without selection of V60I or S162A before the patient initiated therapy (data not shown).

Interestingly, except for the pM41L variant, all patient-derived viruses had a higher RC than the corresponding site-directed mutants (Figure 1). The RC of all protease mutation-harboring patient-derived viruses was higher than the corresponding SDM-viruses, and the RC of pL90M and pI54V + V82A + L90M were even higher than WT. In line with these results, the RC of pK103N and pM41L + T69S + L210E + T215S surpassed the RC of the corresponding SDM-viruses to the level of wild type virus. These observations suggest the presence of compensatory mutations in the genetic backbone of patient-derived viruses at the moment of diagnosis that are able to restore viral RC.

Discussion

In this study we strived to explain the in vivo persistence of the majority of TDRM in patients diagnosed with a drug-resistant HIV-1 variant. We selected patients diagnosed with HIV-1 containing limited profiles of TDRM, which are the most frequently transmitted variants as shown by large epidemiological studies [2],[4]. In our patients, the vast majority of TDRM persisted for at least a year and up to eight years, confirming observations from previous studies that except for M184V/I, TDRM generally persist for longer than one year [10],[13]-[25].

To explore the potential role of viral RC in persistence of TDRM, we investigated the impact of TDRM on the RC. In vitro determination of RC in PBMCs demonstrated that most site-directed mutant viruses harboring 1–2 frequently observed TDRMs had a reduced RC. However, in line with in vivo persistence the majority of patient-derived viruses had a higher RC than the corresponding SDM viruses. This suggests that polymorphisms, which may be present at baseline, can act as compensatory mutations. Our extensive sequence analysis demonstrated limited evolution on polymorphic positions, suggesting that in many transmitted HIV variants harboring TDRM compensatory mutations are already present at diagnosis.

Of the investigated site-directed mutant viruses, T215Y is known to evolve to atypical or partial revertant amino acids. Such alternative amino acids are known to confer limited impact on viral RC [9],[18],[31], which is in line with the observed persistence of revertant and atypical T215 variants in our and other studies [10],[13],[15]-[25].

Interestingly, when present as a SDM in the commonly used lab strain HXB2, K103N decreased the RC in our experiments although this NNRTI-related mutation has been described to have a low impact in several [32]-[34] but not all [35] previous studies. This discrepancy may be due to the use of different assays or differences in replication caused by polymorphisms in lab strains. Indeed, the RC of patient-derived K103N was similar to WT virus, indicating that polymorphisms can restore viral RC. This may explain the in vivo persistence of K103N in our and previous studies [10],[21].

Several papers have described the impact of some drug resistance mutations on the RC of HIV-1 [16],[32],[33],[35]. To our knowledge, the viral RC of frequently observed protease and RT TDRM has never been compared. Our data reveal that site-directed mutations at position 46 in protease have the most severe impact on RC.

Lack of reversion of the TDRM could be explained by a relatively small viral population size resulting in limited evolution. However, the median plasma HIV-RNA level of the included patients is similar to the HIV-RNA generally observed for newly diagnosed patients in the SPREAD programme [3]. Furthermore, although viral evolution was limited, in all transmitted viral variants changes at polymorphic sites were observed, indicating that replication could result in molecular evolution.

Certain resistance mutations such as M46I in protease have been described to decrease recognition of epitopes by certain HLA types [36]. As a result, also the immune system may affect viral evolution and persistence of TDRM. However, the majority of frequently observed TDRM may not impact or can even enhance recognition of epitopes [36],[37] and as such, it is unlikely that the immune system is the major driving force behind persistence of all TDRM.

We previously hypothesized based on an extensive literature study that the lack of reversion is related to the RC of transmitted HIV-1 variants harboring TDRM [9]. The currently described data confirms that TDRM may persist due to a high RC of the transmitted HIV-1 variant. Alternatively, the selection of additional mutations may restore the RC or result in compensatory fixation [30],[38]. This important role of polymorphisms was supported by the differential impact of TDRM in the presence of patient-derived genetic background compared to site-directed mutants. For all but one investigated frequently observed TDRM, in vitro RC of patient-derived virus was higher than the corresponding SDM. A striking example is M46L. Although the single presence of M46L in HXB2 causes a large decrease in viral RC, this defect in RC is largely restored when M46L is present in a patient-derived genetic background.

M41L is one of the most frequently observed TDRM, and is an intriguing example emphasizing the impact of the genetic background on RC. As a single mutation, M41L in the background of wild type virus HXB2 decreased the RC. This decrease was even more pronounced in the genetic background of pM41L, which was specifically selected for this study because of the absence of known compensatory mutations V60I and S162A [30]. In sharp contrast, pM41L + T69S + L210E + T215S, the patient-derived virus with an extensive profile containing a M41L in the presence of the compensatory mutation V60I had a similar RC as wild type virus [30].

In addition, compensatory mutations may be observed outside the target gene of the antiviral compound. It has been demonstrated that mutations in gag may help to compensate the reduced protease activity conferred by resistance mutations in the protease itself [39]. Unfortunately sequencing of gag is usually not included in routine genotyping within Europe, impeding investigation of a potentially compensatory role of gag in this study. For RT, compensatory mutations may also be present in the connection domain [40], which again is not included in routine genoptyping.

For only a subset of patients we had laboratory evidence of recent infection. We cannot exclude that patients were initially infected with a viral variant harboring a more extensive resistance profile and that some of these mutations had reverted before the patients were diagnosed. As such, the observed limited evolution of pol may be a result of viral adaptation before diagnosis or may even have taken place in previous hosts. By using a more sensitive sequence method, we might have been able to increase the detection of TDRM in the included patients [11]. However, we have previously used ultra-deep sequencing to investigate the quasispecies in plasma of patients who were newly diagnosed with an HIV-1 variant harboring a single NRTI-related resistance mutation. In most patients we were unable to detect viral minority variants harboring more extensive resistance profiles in the plasma, which may be suggestive of infection with a circulating HIV-1 variant harboring a limited resistance profile [41]. It is not unlikely that onward transmission of highly stable HIV-1 variants harboring limited resistance profiles greatly contributes to the current epidemic of transmitted drug resistant HIV-1 variants. Indeed, phylogenetic studies have demonstrated that onward transmission by untreated patients is a major source of transmission of drug-resistant HIV-1 [42]-[44].

It is of great clinical importance to be able to distinguish whether transmitted drug resistant HIV-1 variants harbor complex but partially reverted resistance profiles or circulating HIV-1 variants containing limited resistance profiles. For the frequently observed NNRTI-resistance mutation K103N, it is well-known that it causes high levels of resistance against all first generation NNRTIs [45],[46]. Even when K103N is present as minority variant, it can contribute to therapy failure [11]. Fortunately, the recently approved second-generation NNRTIs remain active against HIV-1 harboring a single K103N [47],[48]. In contrast, we have demonstrated that the NRTI-related M41L in RT has limited impact on selection of resistance against currently used NRTIs [49]. M46I/L or L90M as a single TDRM in protease may cause low level resistance to commonly used protease inhibitors such as lopinavir.

Conclusion

In conclusion, we confirmed persistence of the most frequently observed TDRM. All transmitted HIV-1 variants harbored additional polymorphisms, with limited selection of additional mutations. Limited reversion of TDRM is in concordance with the high in vitro RC of patient-derived viruses harboring TDRM. As SDM viruses with the same TDRM as patient-derived viruses have a lower RC in vitro, we propose that polymorphisms that function as compensatory mutations (partially) restoring viral RC explain the in vivo persistence of TDRM. The stability of transmitted drug resistant HIV-1 variants can facilitate onward transmission of these viruses.

Methods

In vivoevolution

Ethics statement

Ethical requirements differ between countries according to national legislation. In countries where a national surveillance system was established, legally no informed consent was needed. In other countries, approval was obtained by the institutional medical ethical review committees. All data were anonymized at national level.

Patients

Patients from four countries participating in the SPREAD-programme (Belgium, Greece, the Netherlands, Slovenia) were included. For all included patients, a baseline genotypic resistance test performed on a plasma sample within three months after diagnosis of HIV-1 infection revealed at least one mutation on a position associated with transmitted drug resistance as described in the mutation list as recommended by the WHO [26]. Patients were included on the basis of sample availability; a base line sample and a sample one year (10–14 months) later. If available, a sample at later time points were included. All included patients were at least 18 years of age and not exposed to antiretroviral therapy during the study period.

Sequence analysis

Genotypic resistance tests were performed by population sequencing of the viral protease and part of reverse transcriptase using commercially available assays or in-house methods covering at least amino acids 4–99 of protease and amino acids 30–249 of RT. All laboratories collaborated in the quality control program of ESAR to ensure high quality genotypic data [3],[4]. HIV-1 subtype was determined using REGA 2.0 [50]. To investigate evolution, the p-distance and the ratio of the proportions of synonymous and nonsynonymous substitutions (dS/dN ratio) were calculated using MEGA 5.05. The p-distance is the proportion of nucleotides between two sequences that has been changed. The dS/dN ratio, a measure of selection pressure [51], was calculated with the Nei-Gojobori method and statistically tested with a Z-test.

In vitrodetermination of replicative capacity

Virus panel

Mutations M46I, M46L and L90M in protease and M41L, M41L + T215Y and K103N in RT were introduced in HXB2 by site-directed mutagenesis using the previously described vector systems CP-MUT and NRT-MUT [52] and the following primers: M46I 5′-GGA AAC CAA AAA TAA TAG GG-3′ (HXB2 nucleotides 2380–2396), M46L 5′-GGA AAC CAA AAC TGA TAG GG-3′ (HXB2 nucleotides 2380–2396), L90M 5′-GAA ATC TGA TGA CTC AGA TTG-3′ (HXB2 nucleotides 2511–2532), M41L 5′-ATT TGT ACA GAG CTG GAA AAG GAA G-3′ (HXB2 nucleotides 2658–2682), K103N 5′-GTT ACT GAT TTG TTC TTT TTT AAC CC-3′ (HXB2 nucleotides 2844–2869), T215Y 5′-TGTCTG GTG TGTAAA GTCCCCACC-3′ (HXB2 nucleotides 3181–3204).

Baseline patient-derived viral protease genes harboring M46I, M46L, L90M or I54V + V82A + L90M or the N-terminus of RT containing M41L, M41L + T69S + L210E + T215S or K103N were introduced into HXB2 using the same vector system [52].

Clones were obtained and sequence analysis was performed to verify resemblance to population sequences. Subsequently, at least three recombinant virus stocks were generated by Lipofectamine 2000 (Invitrogen) transfection of HEK293T cells according to manufacturer’s guidelines. TCID50 was determined by end-point dilution in MT2 cells, demonstrating similar replication in this T cell line in all cases. A random clone was selected and quantified by p24 ELISA (Aalto Bioreagent, Dublin, Ireland) for the RC analysis.

RC analysis

PBMCs were isolated from HIV-seronegative blood donors by Ficoll-Paque density gradient centrifugation and stored in liquid nitrogen until use. To minimize differences between batches caused by variation between donors, each batch of PBMCs consisted of five combined donors. The RC of the virus panel was determined by infecting 5×106 phytohaemagglutinin-stimulated (2 mg/L) donor PBMCs with the equivalent of 40 ng HIV-1 p24 for two hours. Subsequently, cells were washed twice and maintained for 14 days in RPMI1640 with L-glutamine (BioWhittaker), 10% fetal bovine serum (Biochrom AG), 10 mg/L gentamicin (Gibco) and 5 U/ml IL-2. Cell-free supernatant was harvested daily for monitoring of the p24 production. The RC of either the SDM-viruses or the patient-derived viruses was compared to the RC of control viruses (WT, HIV-M184V, −M184I and –M184T). By comparing viruses containing only the mutation(s) or gene of interest in the exact same HIV-WT background, it is possible to determine the impact of these relevant mutation(s) or genes on viral RC. For all viruses, replication curves were performed in four biological replicates divided over two independent experiments. The mean p24 production of two replicates within representative experiments are indicated in Figure 1A-C for protease and 1D-F for RT. Figure 1G represents the median p24 production relative to HIV-WT of all four replicates on day 7 post infection.

Declarations

Acknowledgements

This work was supported by the Aids Fonds Netherlands [grant 2007034] and the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)” [grant agreement n° 223131].

We acknowledge the support of contributors to the SPREAD surveillance programme and members of the European Society for Antiviral Resistance (ESAR). We thank Petra van Ham and Leander Theissen for technical assistance, Daniel Struck from the ESAR-team in Luxembourg for assistance in data management and Antoinet van Kessel for assistance in project coordination.

Authors’ Affiliations

(1)
Department of Virology, Viroscience Lab, Erasmus MC
(2)
Virology, Department of Medical Microbiology, University Medical Center Utrecht
(3)
Institute of Tropical Medicine
(4)
AIDS Reference Laboratory of the Vrije Universiteit Brussel, subunit Universitair Ziekenhuis Brussels
(5)
Internal Medicine and Infectious Diseases, University Medical Center Utrecht
(6)
National Retrovirus Reference Center, Department of Hygiene, Epidemiology and Medical Statistics
(7)
Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana
(8)
On behalf of the SPREAD programme

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