![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Virology, December 2001, p. 11920-11923, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11920-11923.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Partial Restoration of Replication of Simian
Immunodeficiency Virus by Point Mutations in either the Dimerization
Initiation Site (DIS) or Gag Region after Deletion Mutagenesis within
the DIS
Yongjun
Guan,1,
Karidia
Diallo,1,2
Mervi
Detorio,1
James B.
Whitney,1,2
Chen
Liang,1 and
Mark A.
Wainberg1,2,*
McGill AIDS Center, Lady Davis
Institute-Jewish General Hospital, Montreal, Quebec H3T
1E2,1 and Department of Microbiology and
Immunology, McGill University, Montreal, Quebec H3A
2B4,2 Canada
Received 19 April 2001/Accepted 4 September 2001
 |
ABSTRACT |
We used the simian immunodeficiency virus (SIV) molecular clone
SIVmac239 to generate a deletion construct, termed SD2, in which we
eliminated 22 nucleotides at positions +398 to +418 within the putative
dimerization initiation site (DIS) stem. This SD2 deletion severely
impaired viral replication, due to adverse effects on the packaging of
viral genomic RNA, the processing of Gag proteins, and viral protein
patterns. However, long-term culture of SD2 in either C8166 or CEMx174
cells resulted in restoration of replication capacity, due to two
different sets of three compensatory point mutations, located within
both the DIS and Gag regions. In the case of C8166 cells, both a K197R
and a E49K mutation were identified within the capsid (CA) protein and
the p6 protein of Gag, respectively, while the other point mutation
(A423G) was found within the putative DIS loop. In the case of CEMx174
cells, two compensatory mutations were present within the viral
nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G
substitution as observed with C8166 cells. A set of all three mutations
was required in each case for restoration of replication capacity, and
either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.
| |
TEXT |
The 5' untranslated leader sequences
of both simian immunodeficiency virus (SIV) and human immunodeficiency
virus type 1 (HIV-1) possess distinct functional domains that include
elements required for transactivation of transcription, initiation of
reverse transcription, packaging of viral RNA, and integration of the
proviral genome (7). Leader sequences downstream of the U5
region include the major splice donor site and are involved in
encapsidation of full-length viral genomic RNA as well as viral gene
expression (11, 14-19). The secondary structure of the
HIV-1 leader sequence, located downstream of U5, includes four
stem-loop RNA motifs, termed SL1, SL2, SL3, and SL4 (5,
13). Each of the SL1, SL3, and SL4 elements has been shown to be
involved in packaging of viral genomic RNA (7). In
addition, SL1 contains a palindromic loop sequence that is thought to
serve as a dimerization initiation site (DIS) (6, 25).
Mutations within SL1 have been shown to severely diminish viral
infectivity (23).
The 5' leader sequence of SIV has little sequence similarity to that of
HIV-1; however, similar secondary structures are predicted for both
(11, 24). A stem-loop structure that serves as a putative
DIS has been identified in the leader sequences of both SIV and HIV-1.
Our group has previously shown that deletions within the DIS stem-loop
of each of the viruses HIV-1 and SIV severely impaired viral
replication and that this led to impairment of both Gag protein
processing and packaging of viral genomic RNA (11, 12, 19,
20). Reversion to wild-type viral replication has been observed
following deletions within the DIS of HIV-1, and this has been
attributed to a series of four point mutations within distinct Gag
proteins, i.e., matrix (MA), capsid (CA), p2, and nucleocapsid (NC)
(21). In the case of SIV, deletion of presumed DIS
sequences at positions +398 to +418 yielded a construct termed SD2.
This virus was initially impaired in replication ability in C8166
cells; nonetheless, long-term passage led to a restoration of viral
replication that was shown to be due to three point mutations, located
in both the putative DIS (an A423G substitution) and Gag regions (K197R
and G49K) (11). However, the mechanisms whereby these
mutations might contribute to the observed rebound of viral replication
capacity were not characterized.
We have now extended our studies of the reversion of deleted SD2
viruses in both C8166 cells and CEMx174 cells and identified a
different set of compensatory mutations, i.e., Glu18Gln (E18G) and
Gln31Lys (Q31K), both located in the nucleocapsid protein of Gag, and
the same A423G substitution in the putative DIS, responsible for
restored viral growth.
All three point mutations, i.e., A423G, K197R, and E49K, are
required for full replication of the SD2 variant.
Research
involving cell lines, viral stocks, viral replication, viral protein
analysis, and packaging of viral genomic RNA was carried out as
described elsewhere (11). To shed further light on the
role of these mutations in the restoration of replication, a series of
seven SD2 derivatives were generated that represent all of the
combinations of the three point mutations previously identified. These
viruses contained either one, two, or all three of the above-mentioned
mutations, as follows: SD2-A423G; SD2-K197R; SD2-E49K; SD2-A423G,K197R;
SD2-A423G,E49K; SD2-K197R,E49K; and SD2-A423G,K197R,E49K. As shown in
Fig. 1, each of the above-mentioned point
mutations was able to partially compensate for the SD2 deletion in
C8166 cells; however, all three mutations in tandem were required for
full replication capacity, and a previously observed polymorphism, i.e., T310C (12), located upstream of the putative DIS,
was unable to restore even minimal viral replication.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
Growth curves of reverted viruses in C8166 cells.
Compensatory mutations located within both the DIS, i.e., A423G, and
the Gag region, i.e., K197R and E49K, were introduced into the SD2
construct. The resultant constructs were then transfected into COS-7
cells. Equivalent amounts of recovered viruses, based on levels of p27
antigen (10 ng/106 cells), were used to infect C8166 cells,
Viral replication was monitored by reverse transcriptase (RT)
assay of culture fluids. Mock infection denotes exposure of cells to
heat-inactivated wild-type (WT) virus as a negative control.
|
|
Reversion of SD2 virus in CEMx174 cells.
We also passaged the
SD2 virus over multiple generations in the CEMx174 cell line; these
studies led to recovery of a virus with wild-type replication
properties. The viral DNA fragment from the U5 region to the end of the
gag gene of this reverted virus was amplified and cloned as
described previously, and six of these clones (from two independent
series of passages) were sequenced (11) The results show
that the original SD2 deletion had been retained in each case but that
three additional point mutations were also present. One of these was
the same as that previously identified in the aftermath of SD2
reversion in C8166 cells, i.e., A423G, but two distinct mutations
located in the NC region were also identified, i.e., E18Q and Q31K. The
E18Q mutation (Glu
Gln) was located within the first zinc finger
domain of the NC protein, while the Q31K substitution (Gln-Lys) was
found further downstream within NC.
Site-directed mutagenesis was then performed to introduce each of these
NC mutations, alone, together, and in combination with the A423G
substitution, into the SD2 genome. This was performed using the
QuickChange site-directed mutagenesis kit (Stratagene, La Jolla,
Calif.) (11, 21, 22) and the following primer pairs, nc1-1
(5'-GTGTTGGAATTGTGGGAAACAGGGACACTCTGCAAGGC-3') and nc1-2
(5'-GCCTTGCAGAG TG TCCC TG T T TCCCACAAT TCCAACAC - 3') for E18Q and nc2-1
(5'-GCAGAGCCCCAAGAAGAAAGGGATGCTGGAAATGTGG-3') and nc2-2
(5'-CCACATTTCCAGCATCCCTTTCTTCTTGGGGCTCTGC-3') for Q31K. Different combinations of these point mutations were also generated using the same methods. The resultant constructs, termed
SD2-E18Q; SD2-Q31K; SD2-E18Q,Q31K; SD2-A423G,E18Q;
SD2-A423G,Q31K; and SD2-A423G,E18Q,Q31K, were then
transfected into COS-7 cells, and the virus particles thereby recovered
were assayed for viral replication capacity in CEMx174 cells. The
results in Fig. 2 show that various of
these mutations could individually contribute to recovered viral growth but that full restoration of replication capacity required the combination of all three mutations, i.e., SD2-A423G,E18Q,Q31K.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2.
Growth curves of reverted viruses in CEMx174 cells. The
second set of compensatory mutations, located in the DIS, i.e., A423G,
and the NC region, i.e., E18Q and Q31K, were introduced, either alone
(A) or combined (B), into the SD2 construct. The resultant
constructs were transfected into COS-7 cells. Equivalent amounts of
virus thereby recovered were used to infect C8166 cells based on levels
of p27 antigen (10 ng/106 cells). Viral replication was
monitored by reverse transcriptase (RT) assay of culture fluids. Mock
infection denotes exposure of cells to heat-inactivated wild-type (WT)
virus as a negative control.
|
|
Our findings on the involvement of mutations in NC are consistent with
studies of HIV-1 that showed that the NC protein is the major domain
within Gag that recognizes the encapsidation signals present within
leader sequences (2-4). The NC protein contains two zinc
finger motifs that contribute to its specific interactions with viral
RNA (8-10), and our data point to a role for NC in
interactions between Gag and RNA leader sequences. Of course, the p6
protein is also important in the incorporation of viral proteins into
virions and is involved in encapsidation of viral RNA (1, 9,
22). Mechanistic studies are in progress to assess the role of
each of the mutations identified above in restoration of viral replication.
The two sets of compensatory mutations can restore viral
replication equally in each of the cell lines C8166 and CEMx174.
We next tested the infectiousness of viruses containing each of the
above-described sets of mutations in both cell types. Figure
3 demonstrates that the mutations that
had emerged in CEMx174 cells, i.e., A423G, E18Q, and Q31K, were also
able to rescue the SD2 deletion in C8166 cells and that the A423G,
K197R, and E49K mutations rescued viral replication in CEMx174 cells.
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 3.
Replication capacity of wild-type (WT) and mutated
viruses in C8166 cells or CEMx174 cells. Equivalent amounts of virus
were used to infect cells based on levels of p27 antigen (10 ng/106 cells). Viral replication was monitored by reverse
transcriptase (RT) assay of culture fluids. Mock infection denotes
exposure of cells to heat-inactivated wild-type virus as a negative
control. (A) Growth curves of the second set of SD2 reverted viruses in
C8166 cells. (B) Growth curves of the first set of SD2 reverted viruses
in CEMx174 cells.
|
|
Moreover, the infectiousness of SD2 deleted viruses, containing either
of these sets of mutations, but not individual substitutions, was
similar to that of wild-type virus in CEMx174 cells, as determined by
assay of 50% tissue culture infective dose per nanogram of p27 antigen
(results not shown).
Thus, we have shown that both sets of compensatory mutations selected
in these studies were able to act interchangeably to restore viral
replication, regardless of the cell type in which the virus was grown.
Of course, it is conceivable that either the same mutational patterns
or different ones may have been observed in either of the cell lines
studied had either additional replication studies been performed or
different conditions for cell growth been employed. Nonetheless, it is
not trivial that the escape mechanisms for lentiviruses, grown under
conditions of stress as demonstrated here, are apparently not
restricted to single pathways, and this is the first demonstration of
such plasticity on the part of a lentiviral genome.
| |
ACKNOWLEDGMENTS |
This research was supported by grant RO1 AI43878-01 to M.A.W. from
the National Institute of Allergy and Infectious Diseases, National
Institutes of Health.
The CEMx174 cell line was obtained from Peter Cresswell through the
AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH. We thank Maureen Oliveira for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: McGill AIDS
Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote
Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. Phone: (514)
340-8260. Fax: (514) 340-7537. E-mail:
mwainb1{at}po-box.mcgill.ca.
Present address: Division of Clinical Sciences, University of
Toronto, Toronto, Ontario, Canada M5S 1A8.
| |
REFERENCES |
| 1.
|
Accola, M. A.,
A. A. Bukovsky,
M. S. Jones, and H. G. Gottlinger.
1999.
A conserved dileucine-containing motif in p6gag governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIVmac and SIVagm.
J. Virol.
73:9992-9999[Abstract/Free Full Text].
|
| 2.
|
Aldovini, A., and R. A. Young.
1990.
Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.
J. Virol.
64:1920-1926[Abstract/Free Full Text].
|
| 3.
|
Berkowitz, R. D., and S. P. Goff.
1994.
Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein.
Virology
202:233-246[CrossRef][Medline].
|
| 4.
|
Berkowitz, R. D.,
A. Ohagen,
S. Hoglund, and S. P. Goff.
1995.
Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo.
J. Virol.
69:6445-6456[Abstract].
|
| 5.
|
Clever, J.,
C. Sassetti, and T. G. Parslow.
1995.
RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1.
J. Virol.
69:2101-2109[Abstract].
|
| 6.
|
Clever, J. L.,
M. L. Wong, and T. G. Parslow.
1996.
Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA.
J. Virol.
70:5902-5908[Abstract].
|
| 7.
|
Coffin, J. M.,
S. H. Hughes, and H. E. Varmus.
1997.
Retroviruses.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 8.
|
Dannull, J.,
A. Surovoy,
G. Jung, and K. Moelling.
1994.
Specific binding of HIV-1 nucleocapsid protein to PSIRNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.
EMBO J.
13:1525-1533[Medline].
|
| 9.
|
Dettenhofer, M., and X.-F. Yu.
1999.
Proline residues in human immunodeficiency virus type 1 p6Gag exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins.
J. Virol.
73:4696-4704[Abstract/Free Full Text].
|
| 10.
|
Dorfman, T.,
J. Luban,
S. P. Goff,
W. A. Haseltine, and H. G. Gottlinger.
1993.
Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein.
J. Virol.
67:6159-6169[Abstract/Free Full Text].
|
| 11.
|
Guan, Y.,
J. B. Whitney,
K. Diallo, and M. A. Wainberg.
2000.
Leader sequences downstream of the primer binding site are important for efficient replication of simian immunodeficiency virus.
J. Virol.
74:8854-8860[Abstract/Free Full Text].
|
| 12.
|
Guan, Y.,
J. B. Whitney,
C. Liang, and M. A. Wainberg.
2001.
Novel live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences.
J. Virol.
75:2776-2785[Abstract/Free Full Text].
|
| 13.
|
Harrison, G. P., and A. M. L. Lever.
1992.
The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure.
J. Virol.
66:4144-4153[Abstract/Free Full Text].
|
| 14.
|
Harrison, G. P.,
G. Miele,
E. Hunter, and A. M. L. Lever.
1998.
Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line.
J. Virol.
72:5886-5896[Abstract/Free Full Text].
|
| 15.
|
Kaye, J. F., and A. M. L. Lever.
1999.
Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation.
J. Virol.
73:3023-3031[Abstract/Free Full Text].
|
| 16.
|
Kim, H. J.,
K. Lee, and J. J. O'Rear.
1994.
A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA.
Virology
198:336-340[CrossRef][Medline].
|
| 17.
|
Lenz, C.,
A. Scheid, and H. Schaal.
1997.
Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression.
J. Virol.
71:2757-2764[Abstract].
|
| 18.
|
Lever, A.,
H. Gottlinger,
W. Haseltine, and J. Sodroski.
1989.
Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions.
J. Virol.
63:4085-4087[Abstract/Free Full Text].
|
| 19.
|
Liang, C.,
X. Li,
Y. Quan,
M. Langhrea,
L. Kleiman,
J. Hiscott, and M. A. Wainberg.
1997.
Sequence elements downstream of human immunodeficiency virus type 1 long terminal repeat are required for efficient viral gene transcription.
J. Mol. Biol.
272:167-177[CrossRef][Medline].
|
| 20.
|
Liang, C.,
L. Rong,
E. Cherry,
L. Kleiman,
M. Laughrea, and M. A. Wainberg.
1999.
Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins.
J. Virol.
73:6147-6151[Abstract/Free Full Text].
|
| 21.
|
Liang, C.,
L. Rong,
Y. Quan,
M. Laughrea,
L. Kleiman, and M. A. Wainberg.
1999.
Mutations within four distinct Gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site.
J. Virol.
73:7014-7020[Abstract/Free Full Text].
|
| 22.
|
Ott, D. E.,
E. N. Chertova,
L. K. Busch,
L. V. Coren,
T. D. Gagliardi, and D. G. Johnson.
1999.
Mutational analysis of the hydrophobic tail of the human immunodeficiency virus type 1 p6Gag protein produces a mutant that fails to package its envelope protein.
J. Virol.
73:19-28[Abstract/Free Full Text].
|
| 23.
|
Poon, D. T. K.,
J. Wu, and A. Aldovini.
1996.
Changed amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.
J. Virol.
70:6607-6616[Abstract/Free Full Text].
|
| 24.
|
Rizvi, T. A., and A. T. Panganiban.
1993.
Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.
J. Virol.
67:2681-2688[Abstract/Free Full Text].
|
| 25.
|
Skripkin, E.,
J. C. Pailart,
R. Marquet,
B. Ehresmann, and C. Ehresmann.
1994.
Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.
Proc. Natl. Acad. Sci. USA
91:4945-4949[Abstract/Free Full Text].
|
Journal of Virology, December 2001, p. 11920-11923, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11920-11923.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Whitney, J. B., Oliveira, M., Detorio, M., Guan, Y., Wainberg, M. A.
(2002). The M184V Mutation in Reverse Transcriptase Can Delay Reversion of Attenuated Variants of Simian Immunodeficiency Virus. J. Virol.
76: 8958-8962
[Abstract]
[Full Text]
Top
Abstract
Text
