Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes.

Abstract

APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5'-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.

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JOURNAL OF VIROLOGY, May 2005, p. 5870–5874
0022-538X/05/$08.00?0
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 9
doi:10.1128/JVI.79.9.5870–5874.2005
Viral RNA Is Required for the Association of APOBEC3G with
Human Immunodeficiency Virus Type 1
Nucleoprotein Complexes
Mohammad A. Khan,1Sandra Kao,1Eri Miyagi,1Hiroaki Takeuchi,1Ritu Goila-Gaur,1
Sandrine Opi,1Clay L. Gipson,2Tristram G. Parslow,2Hinh Ly,2
and Klaus Strebel1*
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda,
Maryland 20892-0460,1and Emory University, Department of Pathology and Laboratory Medicine,
Experimental Pathology Division, Atlanta, Georgia 303222
Received 10 December 2004/Accepted 21 December 2004
APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus
type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1
nucleoprotein complexes (NPC). A target sequence located within the 5?-untranslated region of the HIV-1 RNA
was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence
normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging
and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by
viral RNA.
Replication of human immunodeficiency virus type 1
(HIV-1) in primary cells is dependent on the expression of Vif
protein, which counteracts the activity of the host cytidine
deaminases APOBEC3G (APO3G) and APOBEC3F (4, 25,
29, 32). In the absence of Vif, APO3G is incorporated into
virus particles (11, 16, 19, 20, 26, 27, 30), resulting in hyper-
mutation of the viral genome (19) or degradation of mutated
cDNA (14, 18, 31) via a DNA repair mechanism (reviewed in
references 3 and 12). Interestingly, human APO3G is not only
packaged into human immunodeficiency viruses but also incor-
porated into simian immunodeficiency viruses and murine leu-
kemia virus (9, 18, 19). Packaging of APO3G into such diverse
viruses suggests that it either is a relatively nonspecific process
or involves signals shared by these viruses. APO3G can bind
RNA in vitro (10). Indeed, several reports have noted that the
presence of viral RNA enhanced APO3G encapsidation (28);
however, others (17, 23) suggested that viral RNA was not
essential for APO3G packaging (2, 5, 8, 17, 23, 28).
To further study the role of viral RNA in the packaging of
APO3G into HIV-1 virions, we first compared the packaging
of APO3G into either the wild-type infectious NL4-3 virus or
a helper virus (C-Help) whose RNA genome is not packaged
due to a 33-base deletion in the putative RNA packaging signal
(21). Virus stocks were prepared by transient cotransfection of
HeLa cells with either the pNL4-3 plasmid (1) or the C-Help
vector DNA and the APO3G-expressing plasmid pcDNA-
APO3G (11). Viruses were collected 48 h after transfection
and purified by two rounds of sucrose gradient centrifugation.
Cell lysates and concentrated virus preparations were analyzed
by immunoblotting (Fig. 1A). We found that packaging of
APO3G into helper virus was reduced by ?3.5-fold compared
to packaging into NL4-3 virus (Fig. 1B). Thus, viral RNA
contributes to the specific packaging of APO3G into HIV-1
virions, consistent with data reported by Svarovskaia et al. (28).
If encapsidation of APO3G and viral RNA are linked, the
APO3G packaging defect observed with the helper virus (Fig.
1A and B) should be overcome by the coexpression of pack-
aging-competent vector-derived RNA. To test this hypothesis,
several packaging vectors were constructed based on the len-
tiviral packaging vector pHR?CMVGFP (15). This vector con-
tains both HIV-1 long terminal repeats (LTRs), the 5?-untrans-
lated region, 350 nucleotides of gag, and 880 nucleotides of an
env segment encompassing the Rev-responsive element (RRE)
region. Vector ?C (Fig. 1C) is similar to pHR?CMVGFP ex-
cept for a 650-bp deletion in the cytomegalovirus promoter
region, which prevents expression of the green fluorescent
protein (GFP) reporter gene. This deletion is present in all
other constructs tested here. Vector ?L lacks the LTR regions
and is under the transcriptional control of an SV40 promoter.
Vector ?R lacks the RRE-containing env sequence and con-
tains a codon-optimized gag coding sequence (24). A vector
containing a deletion of both LTRs and RRE (?L/R) was also
constructed (Fig. 1C). As illustrated in Fig. 1D, coexpression of
the packaging vector (?C) resulted in a sixfold increase in the
packaging of APO3G compared to the helper virus alone.
Similarly, cotransfection of the ?L, ?R, and ?L/R variant
constructs increased packaging of APO3G by six- to sevenfold.
These results suggest that the 5?-untranslated region and the
first 350 nucleotides of gag were sufficient for APO3G encap-
sidation into HIV-1 virions.
The 5?-untranslated region contains numerous secondary
structures required for virus replication (Fig. 2A) (reviewed
in reference 22). To further map the sequences and RNA
secondary structures required for APO3G packaging, we
* Corresponding author. Mailing address: NIH, NIAID, 4/312, 4
Center Drive, MSC 0460, Bethesda, MD 20892-0460. Phone: (301)
496-3132. Fax: (301) 402-0226. E-mail: kstrebel@nih.gov.
5870

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tested two stem-loop mutants, mS.1 and mS.3 (Fig. 2A),
located in a region that has been shown to be important for
viral RNA dimerization and encapsidation. The sequences
located on both strands of the individual helical structures
of stem-loop 1 (SL1) or stem-loop 3 (SL3) were swapped in
mS.1 and mS.3 mutants, respectively, thus changing the pri-
mary sequences of the stems without perturbing formation
of the hairpin structures. These mutants were selected be-
cause they package viral RNA at or near wild-type levels and
were found to produce fully infectious viruses (7). Reverse
transcription (RT)-PCR analysis showed similar levels of
virus-associated RNA in the mS.1 and mS.3 viruses while
C-Help virus did not contain detectable levels of viral RNA
(Fig. 2B). Packaging of APO3G into mS.1 and mS.3 parti-
cles was analyzed by immunoblotting (Fig. 2C). While
APO3G was expressed at similar levels intracellularly (Fig.
2C, lanes 1 and 2), APO3G packaging into mS.1 was re-
duced by ?3-fold compared to mS.3 virus (Fig. 2C, compare
lanes 3 and 4). While our data do not rule out the possible
involvement of other regions in the 5?-untranslated region,
they clearly identify SL1 as a region important for packaging
of APO3G into HIV-1 virions and hence strongly argue for
a specific involvement of viral RNA in APO3G packaging.
We have previously established that components of the viral
core are resistant to detergent extraction, whereas other viral
components, such as matrix (MA) or envelope protein, are
detergent sensitive and can be separated from core-associated
proteins by sucrose step gradient centrifugation (13). In this
FIG. 1. Virus incorporation of APO3G protein requires packaging of viral RNA. HeLa cells were co-transfected with either the full-length
molecular clone pNL4-3 (WT) or a helper virus construct (C-Help) along with pcDNA-APO3G vector DNA. Cells and virus-containing
supernatants were harvested 48 h posttransfection. (A) Whole-cell lysates and purified, concentrated virus preparations were analyzed by
immunoblotting using an anti-APO3G antibody (APO3G) or an HIV-positive patient serum (CA). (B) Packaging of APO3G into virions was
quantified by densitometric analysis of the bands shown in panel A. Results were corrected for variations in CA protein. APO3G levels in NL4-3
preparations were defined as 100%. (C) Schematic presentation of the modifications introduced into the pHR?CMVGFP packaging vector. Vector
?C contains both HIV-1 LTRs, the 5?-untranslated region plus the first 350 nucleotides of the gag gene, an 880-nucleotide fragment containing
the HIV-1 RRE element, and a GFP expression cassette. This vector is identical to pHR?CMVGFP except for a deletion in the cytomegalovirus
promoter to prevent GFP protein expression. Variants lacking the LTR regions (?L), the RRE domain (?R), or both (?L/R) are described in the
text. SV40 and AAA symbolize simian virus 40 promoter and terminator elements, respectively. SD, splice donor site; CMV, cytomegalovirus; SA,
splice acceptor site; Opt. Gag, codon-optimized Gag. (D) Helper virus (C-Help) DNA was transfected into HeLa cells together with either an
empty vector (?) or one of the packaging vectors shown in panel C. Cells and viruses were harvested 48 h after transfection. Packaging of APO3G
was assessed by immunoblotting as described in the legend to panel A, and the level of APO3G packaging was quantified as described in the legend
to panel B. APO3G packaging efficiency is expressed as fold increase over the level observed with helper virus alone (?). Values were corrected
for variations in CA protein. Error bars reflect the standard deviations calculated from three independent experiments.
VOL. 79, 2005NOTES5871

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assay, intact viruses accumulate at the 20%/60% interphase of
the step gradient column (Fig. 3B, S3) as evidenced by the
enrichment of CA and MA proteins in the S3 fraction (Fig. 3A,
lane 3). In contrast, detergent treatment resulted in the parti-
tioning of MA into the soluble S1 fraction (Fig. 3A, lane 4). As
observed previously (13), CA remained partially resistant to
detergent treatment. Overall, the detergent sensitivity of these
viral components was very similar for all samples tested. Inter-
estingly, NL4-3-associated APO3G was largely resistant to de-
tergent extraction (Fig. 3A). In fact, ?70% of the virus-asso-
ciated APO3G copurified with the viral core fraction S3 (Fig.
3C). These results suggest for the first time that APO3G is
packaged into virus particles as a stable complex with the viral
core. Similar results were observed for Vif-defective NL4-3
(Fig. 3D). Thus, the presence or absence of Vif did not affect
the relative affinity of APO3G to the viral core but merely
altered the absolute amounts of virus-associated APO3G. In
contrast, detergent treatment of C-Help virus extracted about
two-thirds of virus-associated APO3G from the core fraction
(Fig. 3E). Similarly, APO3G packaged into mS.1 particles was
highly detergent sensitive (Fig. 3F). Despite the presence of
viral RNA, APO3G association with the mS.1 core was signif-
icantly reduced, suggesting an important role for the sequences
surrounding stem-loop 1 in core association of APO3G. These
biochemical studies therefore support the genetic data pro-
vided in Fig. 2 and strongly argue for a role of viral RNA in
APO3G packaging.
While our data clearly demonstrate that APO3G packaging
into virus particles is enhanced by viral RNA packaging, the
presence of viral RNA per se is not absolutely essential as low
levels of APO3G are packaged even in the absence of viral
RNA (Fig. 1). These results are consistent with previous stud-
ies demonstrating that APO3G can be packaged into viruslike
particles in the absence of viral RNA through an interaction
with the viral NC protein (5, 8, 17, 23, 28). Although APO3G
has RNA binding properties, it is conceivable that APO3G
recognizes a unique RNA-protein complex that forms during
viral assembly as part of the RNA dimerization/encapsidation
process that has been shown to involve the SL1 hairpin as well
as other hairpin structures and sequences (7). Such a mecha-
nism could explain the efficient packaging of APO3G into
diverse viruses, such as HIV-2, simian immunodeficiency virus,
or murine leukemia virus and the sensitivity of APO3G pack-
aging to mutations in NC protein, which has been shown to
participate in both dimerization and encapsidation of viral
RNA (22).
FIG. 2. The SL1 hairpin structure located in the 5?-untranslated region is important for APO3G packaging. (A) Schematic representation of
the RNA structure predicted for the 5?-untranslated region (left panel) (6). TAR, transactivation response region; PBS, primer binding site; SD,
splice donor site. The right panel depicts the nucleotide changes introduced into the stem regions of SL1 and SL3 in the mS.1 and mS.3 mutants,
respectively. wt, wild type. (B) Packaging of viral RNA was analyzed by RT-PCR of the NL4-3mS.1 and NL4-3mS.3 virus stocks using a Vif-specific
primer set. Helper virus (pC-Help) and NL4-3 virus stocks were analyzed in parallel. All virus preparations were normalized by RT activity.
(C) HeLa cells were transfected with plasmids encoding the variants carrying mutations in stem-loop 1 (mS.1) or stem-loop 3 (mS.3) together with
the APO3G expression vector. Cells and virus-containing supernatants were collected 24 h after transfection. Cell lysates and purified, concen-
trated viral pellets were analyzed by immunoblotting as described for Fig. 1.
5872 NOTES J. VIROL.

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We thank George Pavlakis and Antonio Valentin for the codon-
optimized Gag expression vector p55BM1-10PO-2SD?. We thank
Xiao-Fang Yu and Bryan Cullen for sharing unpublished data. We
gratefully acknowledge Alicia Buckler-White and Ron Plishka for oli-
gonucleotide synthesis and sequence analysis.
Part of this work was supported by a grant from the NIH Intramural
AIDS Targeted Antiviral Program to K.S.
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