Differential Effects of Human Immunodeficiency Virus Type 1 Capsid

1

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and 1

Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and 2

Specificity of Viral DNA Integration 3

4

Yasuhiro Koh,1† Xiaolin Wu,2† Andrea L. Ferris,3 Kenneth A. Matreyek,1 Steven J. 5

Smith,3 KyeongEun Lee,3 Vineet N. KewalRamani,3 Stephen H. Hughes,3 and Alan 6

Engelman1* 7

8

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and 9

Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215,1 10

Laboratory of Molecular Technology, SAIC-Frederick Inc., Frederick National Laboratory 11

for Cancer Research, Frederick, Maryland 217022, and HIV Drug Resistance Program, 12

Frederick National Laboratory for Cancer Research, Frederick, Maryland 217023 13

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Running title: Viral and Cellular Factors of HIV-1 DNA Integration 15

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Abstract word count: 200; Total word count: 5,611 17

18

† Y.K. and X.W. contributed equally to this work. 19

*Corresponding author. Mailing address: Department of Cancer Immunology and AIDS, 20

Dana-Farber Cancer Institute, 450 Brookline Ave., CLS-1010, Boston, MA 02215. 21

Phone: (617) 632-4361. Fax: (617) 632-4338. E-mail: 22

alan_engelman(at)dfci.harvard.edu 23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.01148-12 JVI Accepts, published online ahead of print on 24 October 2012

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Retroviruses integrate into cellular DNA non-randomly. Lentiviruses such as 24

human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes 25

and gene-enriched, transcriptionally active regions of chromosomes. The 26

interaction between lentiviral integrase and the cellular protein LEDGF/p75 27

underlies the targeting of gene bodies, whereas recent research has highlighted 28

roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral 29

nuclear import, including transportin 3 and nucleoporin (NUP) 358, in the 30

targeting of gene dense regions of chromosomes. Here, we show that CA 31

mutations, which include the substitution of Asp for Asn74 (N74D), significantly 32

reduce the dependency of HIV-1 on LEDGF/p75 during infection, and that this 33

difference correlates with the efficiency of viral DNA integration. The distribution 34

of integration sites mapped by Illumina sequencing confirms that the N74D 35

mutation reduces integration into gene-rich regions of chromosomes and gene 36

bodies, and reveals previously unrecognized roles for NUP153, another HIV-1 co-37

factor implicated in viral nuclear import, as well as LEDGF/p75, in the targeting of 38

the viral preintegration complex to gene-dense regions of chromatin. A role for 39

the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during 40

infection highlights a connection between the viral capsid and chromosomal DNA 41

integration. 42

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Reverse transcription, which converts the single-stranded RNA genome into linear 44

double-stranded DNA, and the subsequent integration of this DNA molecule into a host 45

cell chromosome, are key steps in the early phase of the human immunodeficiency 46

virus type 1 (HIV-1) replication cycle. The viral enzymes reverse transcriptase (RT) and 47

integrase (IN), along with the genomic RNA, enter the cell enclosed in a cone-shaped 48

shell or core comprised of the viral capsid (CA) protein, and reverse transcription is 49

linked to the partial dissolution of the core through a process known as uncoating (27). 50

3' processing of the viral DNA ends by IN converts the reverse transcription complex 51

into the preintegration complex (PIC) (10, 41). The HIV-1 PIC is actively imported into 52

the nucleus (6, 28) through nuclear pore complexes (NPCs). In the nucleus, the PICs 53

locate to sites of chromosomal DNA for IN-mediated integration. See reference (17) for 54

a recent overview of retroviral reverse transcription, nuclear import, and integration. 55

Retroviral integration is non-random, and different viruses have different 56

integration specificities [reviewed in ref. (7)]. Local DNA sequence preferences, which 57

typically consist of ~12 bp palindromes, have a modest effect on integration site 58

selection (16, 26, 61). PICs also sense certain aspects of higher-order chromatin 59

structure during integration. Lentiviruses, which include HIV-1, target the bodies of 60

active genes, and favor transcriptionally active gene-dense regions of chromosomes 61

(50). The integration of Moloney murine leukemia virus (MLV), a gammaretrovirus, also 62

favors active gene-dense regions, though to a lesser extent than HIV-1 (46). In contrast 63

to the lentiviruses, MLV preferentially integrates into promoter regions and affiliated 64

CpG islands rather than the internal regions of genes (60). Viruses derived from other 65

retroviral genera, including alpharetroviruses (42, 44), deltaretroviruses (16), and 66

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spumaviruses (45, 54), have less specificity for chromatin structural features than 67

lentiviruses or MLV, showing weak preferences for gene promoter regions. 68

Although the mechanisms by which most retroviruses target chromatin during 69

integration are unknown, the binding of lentiviral IN protein to the host binding protein 70

lens epithelium-derived growth factor (LEDGF)/transcriptional co-activator p75 in large 71

part defines the preference of this genus for the bodies of active genes. Cells depleted 72

for LEDGF/p75 expression by RNA interference (RNAi) (34, 56) or gene knockout (39, 73

49, 51) support significantly lower overall HIV-1 integration than controls. The 74

proviruses that do form under these conditions are found much less frequently in active 75

genes (12, 39, 49, 51) with an associated rise in integrations near promoters and 76

affiliated CpG islands (39, 49, 51). LEDGF/p75 has a C-terminally located IN-binding 77

domain (IBD) (11, 57) and N-terminal elements that bind chromatin (36, 55). Novel 78

fusion proteins comprised of the LEDGF/p75 IBD and foreign chromatin binding 79

domains redirect HIV-1 to integrate at positions where the heterologous binding partner 80

preferentially binds, demonstrating a direct role for the host factor in targeting lentiviral 81

integration (20, 23, 52). Neither the preference for the local DNA sequence at the site of 82

HIV-1 integration (39, 49, 51), nor the preference for gene-dense regions of 83

chromosomes (39, 46), seem to be influenced by LEDGF/p75. In vitro experiments with 84

prototype foamy virus showed that mutations in IN affected the preference for local DNA 85

sequence at the site of integration (38). This result suggests that the local target site 86

sequence preferences of other retroviruses, including HIV-1, might also be dictated by 87

IN. Recent work has suggested that viral and cellular factors implicated in PIC nuclear 88

import, including CA (48, 62) and the host proteins transportin (TNPO) 3 and 89

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nucleoporin (NUP) 358 (46), play a role in determining the preference of HIV-1 to target 90

gene-dense regions of chromosomes during integration. 91

Work with MLV/HIV-1 (MHIV) chimeric viruses that contain MLV proteins in place 92

of their HIV-1 counterparts first showed that Gag structural determinants can play a role 93

in HIV-1 integration targeting (33, 46). From the N-terminus, HIV-1 Gag is composed of 94

matrix (MA), CA, spacer peptide (SP) 1, nucleocapsid (NC), SP2, and p6 proteins 95

whereas MLV is composed of MA, p12, CA, and NC (Fig. 1A). The MHIV-mMA12CA 96

chimera, which has MLV MA, p12, and CA in place of HIV-1 MA and CA (Fig. 1A), 97

integrated into genes at a frequency that was intermediate to HIV-1 and MLV (33) and, 98

in contrast to the parental viruses, did not favor gene dense regions of chromatin (46). 99

The gene density targeting profile of MHIV-mMA12CA was similar to what was seen 100

with HIV-1 when either TNPO3 or NUP358 was depleted from HeLa cells by RNAi (46). 101

Work with additional MHIV chimerae, including MHIV-mMA12 (Fig. 1A), and with CA 102

missense mutants, including N74D, revealed CA as the dominant genetic determinant 103

of TNPO3 (14, 31, 32, 66), NUP358 (32, 48), and NUP153 (32, 40, 48) dependency 104

during HIV-1 infection. The N74D mutation, in particular, ablated integration targeting of 105

the PIC to gene dense regions of chromosomes (48), highlighting a link between PIC 106

nuclear import, CA, and integration targeting. Here, we show that CA mutations alter the 107

dependency of HIV-1 on LEDGF/p75, and that the IN-binding host factor concordantly 108

influences the targeting of HIV-1 PICs to gene dense regions of chromosomes. Our 109

results furthermore reveal a subsidiary role for NUP153 in the targeting of 110

transcriptionally active regions of chromosomes during HIV-1 integration. 111

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MATERIALS AND METHODS 113

Plasmid constructs. Plasmids that encode single-round luciferase reporter viruses 114

include MLV/HIVLAI chimerae (62, 63), pNLX.Luc(R-) (37), and pHI-Luc (43). The 115

backbone of pNLX.Luc(R-) was modified by BstEII/XmaI digestion, the ends were 116

repaired with the Klenow fragment of Escherichia coli DNA polymerase, and re-ligated 117

to yield pNLX.Luc(R-)ΔAvrII. HIV-1 CA mutations were generated by site-directed 118

mutagenesis of the HIV-1NL4-3-based packaging construct pHP-dI-N/A (9, 40) or 119

pNLX.Luc(R-)ΔAvrII. The pN/N.Luc(R-) plasmid expressed the D64N/D116N IN active 120

site mutant virus, and the vesicular stomatitis virus G (VSV-G) glycoprotein was 121

expressed from pCG-VSV-G (37). Sequencing was used to show that the PCR-mutated 122

DNAs carried only the desired mutations. 123

Cells, viruses, and infections. HEK293T, HeLa, and mouse embryo fibroblast 124

(MEF) cells were propagated in Dulbecco’s modified Eagle medium supplemented to 125

contain 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. There 126

were two matched pairs of Psip1 knockout MEFs: the E1(f/+) control and the E2(-/-) 127

knockout, and the E6(f/f) control and the E6(-/-) knockout (51). 128

Single round viruses were pseudotyped with the VSV-G envelope following 129

transient transfection of HEK293T cells with two or three different plasmids as described 130

previously (40). The levels of virus production were monitored using a 32P-based assay 131

for exogenous RT activity, and MEFs (1.6 x 104/well) seeded 24 h before infection in 48-132

well plates were infected in duplicate with 2 x 105 RT counts per minute (cpm) for 24 h 133

to determine single round viral infectivities. At 48 h postinfection (hpi), cells were 134

processed for duplicate luciferase assays, and results were expressed as relative light 135

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units (RLU) normalized to the concentration of the total protein in the cell extract (40). 136

MHIV virus inocula were adjusted so that all infections were within approximately 2 log 137

RLU/µg of control MEFs. Where applicable, cyclosporine (Sigma-Aldrich, St. Louis, MO) 138

was added at the time of infection to the final concentration of 5 µM. Normalized 139

infectivity data were log transformed, and statistical analyses were performed by paired 140

two-tailed Student’s t test. 141

Quantitative (Q)-PCR. MEFs (1.6 x 105/well) seeded 24 h before infection in 6-142

well plates were incubated with 2 x 107 RT-cpm of DNase-treated wild type (WT) or CA 143

mutant virus for 2 h, after which the cells were washed with phosphate-buffered saline 144

and replated into separate wells of a 12-well plate. DNA was extracted from the infected 145

cells at various times using the QIAamp DNA Minikit (Qiagen, Valencia, CA), and the 146

DNA samples were analyzed in duplicate or triplicate by Q-PCR to determine the total 147

amount of HIV-1 DNA at 7 hpi. The amount of integrated proviruses was determined at 148

48 hpi using nested BBL-PCR essentially as described previously (40, 51). DNA was 149

prepared from parallel infections conducted in the presence 25 µM azidothymidine 150

(AZT) to determine the background residual transfected plasmid DNA in the Q-PCR 151

assay. This background was subtracted from the experimental values (AZT-treated 152

samples averaged 1.8% of the total and 7.7% of the BBL-PCR peak values). Plasmid 153

DNA [pNLX.Luc(R-)] was used to generate the HIV-1 DNA standard curve, and a 154

polyclonal population of E6(f/f) MEFs infected at a relatively low multiplicity of infection 155

with an HIV-1-based pHI-puro single-round virus and selected for 3 weeks in 2 µg/ml 156

puromycin was used to prepare the cellular DNA for the integration standard (40). 157

Twofold dilutions of this DNA were mixed with DNA prepared from uninfected E6(f/f) 158

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cells to prepare the standard curve. Q-PCR primers and probes for total HIV-1 detection 159

have been described (40). BBL-PCR primers and probes have also been described (51) 160

except that AE3014 and AE3013 replaced first and second round primers AE2257 and 161

AE989, respectively, to increase the specificity of the amplification (19). 162

Integration site libraries. HeLa cells (2.5 x 104) were transfected in 48 well 163

plates with 40 nM short-interfering (si) control or NUP153-targeting RNA #1 (40), pooled 164

(2.5 x 106 cells), and infected 3 d later with 2.5 x 108 RT cpm of WT or N74D mutant 165

virus derived from pNLX.Luc(R-)ΔAvrII. After 2 d, the cells were harvested and DNA 166

was purified as described above. The methods used to prepare the DNA fragments, and 167

amplify and sequence the integration sites, are similar to those described by Gillet et al 168

(24). Briefly, 5 µg of the DNA was sheared into ~300-500 bp fragments by adaptive 169

focused acoustics (Covaris Inc., Woburn, MA) and purified using AMPure XP magnetic 170

beads (Beckman Coulter Genomics, Danvers, MA). The sheared DNA was end-171

repaired, dA-tailed, and ligated to a double-stranded DNA linker as described in 172

Illumina’s sequencing protocol. Both the first and the second round amplifications were 173

done in multiple aliquoted reactions to increase the diversity of the integration site 174

libraries. The resulting PCR products were sequenced using standard chemistry on an 175

Illumina GA2x machine. Sequences that contained the 3'-processed viral DNA through 176

the canonical CA dinucleotide on one end and the ligated linker on the opposing end, 177

and matched a genomic location at >95% identity for a stretch of minimally 20 bp, were 178

counted as HIV-1 integration sites. The genomic sequences were parsed for features 179

such as genes, CpG islands, and transcriptional activity as previously described (20). 180

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Integration site libraries prepared from HIV-1-infected E1(f/+) control and E2(-/-) 181

knockout MEFs (51) were also analyzed by Illumina sequencing. 182

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RESULTS AND DISCUSSION 184

MLV CA confers loss of LEDGF/p75 dependency during HIV-1 infection. 185

LEDGF/p75, which binds directly to lentiviral IN proteins, is an important cellular 186

cofactor for HIV-1 integration [reviewed in (18) and (47)]. Due to its central role in 187

determining the distribution of integrated proviruses in chromosomes, we hypothesized 188

that HIV-1-based constructs that carry changes in proteins other than IN, and have 189

altered integration site preferences, might also have altered infectivity profiles in cells 190

depleted for LEDGF/p75. The MHIV-mMA12CA chimeric virus, which carries the section 191

of the MLV gag gene that encodes MA, p12, and CA (Fig. 1A), is one such virus, as its 192

integration site distribution differed significantly from each of its parents (33, 46). MEF 193

cell lines in which the Psip1 gene (51), which encodes LEDGF/p75 (22), was inactivated 194

were challenged with MHIV-mMA12CA and both parental viruses. Two different pairs of 195

MEF cell lines, E1(f/+), which expresses the LEDGF/p75 protein and the littermate 196

matched E2(-/-) knockout, as well as the LEDGF/p75 expressing cell line E6(f/f) and its 197

matched E6(-/-) knockout, were used in the initial infectivity/integration experiments. 198

Additional chimeric viruses that contained MLV IN either in the HIV-1 (MHIV-mIN) or 199

MHIV-mMA12CA (MHIV-mMA12CAmIN) background, or carried MLV MA and p12 in 200

place of HIV-1 MA (MHIV-mMA12) (62, 63), were included to determine the 201

contributions of these Gag determinants as well as IN under these experimental 202

conditions (Fig. 1A). Because the infectivities of some of the chimeric viruses, especially 203

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those that express the MLV IN protein, are significantly lower than HIV-1 (62, 63), 204

preliminary experiments were conducted to define the levels of the viral inocula that 205

infected the LEDGF/p75-expressing control cells within approximately 2 orders of 206

magnitude of WT HIV-1. Appropriate volumes of the viral stocks were then used to 207

infect matched pairs of LEDGF/p75-expressing and knockout cells. The resulting 208

absolute levels of viral infection, which are plotted with respect to the level of WT HIV-1 209

infection of LEDGF/p75-expressing cells (Fig. 1B and 1C, left panels), were re-graphed 210

as relative viral titer (percent infectivity in knockout cells as compared to the matched 211

LEDGF/p75-expressing cell line; right panels) to simplify the interpretation of which 212

mutation(s) alter the dependency on LEDGF/p75. 213

As previously shown (51), HIV-1 infectivity was significantly reduced by the Psip1 214

knockout, and MLV infectivity was either unaffected [in the E1(f/+) and E2(-/-) cell pair] 215

or affected to a much smaller extent than HIV-1 infectivity (Fig. 1B and 1C, left panels; 216

compare white and black bars). The enhanced HIV-1 infectivity defect in the E6(-/-) 217

knockout cells (Fig. 1C) as compared to E2(-/-) knockout cells correlates with a viral-218

specific transcriptional defect in the E6(-/-) cells (Y.K. and A.E., unpublished 219

observations; also discussed below). The relative titer of the MHIV-mMA12 virus was 220

very similar to HIV-1LAI in both sets of cells, suggesting that the HIV-1 MA protein plays 221

no significant role in LEDGF/p75 dependency. LEDGF/p75 does not interact with or 222

bind to MLV IN (8, 35) and as expected, the relative titers of MHIV-mIN and MLV were 223

not significantly altered by the Psip1 knockout (Fig. 1B and 1C). The infectivity of MHIV-224

mMA12CA, which expresses HIV-1 IN and MLV MA and CA, was noticeably less 225

dependent on LEDGF/p75 than either HIV-1LAI or MHIV-mMA12 (Fig. 1B and 1C). This 226

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reduced dependency of MHIV-mMA12CA on LEDGF/p75 was seen over a 100-fold 227

range in the virus inoculum (data not shown). However, we note that the MHIV-228

mMA12CA chimera was significantly more dependent on LEDGF/p75 than was MHIV-229

mIN, and that the addition of MLV IN to MHIV-mMA12CA caused significant further 230

reductions in the dependency of the Gag chimera virus on the integration co-factor (Fig. 231

1B and 1C). These data confirm the primary role of IN in the dependency of HIV-1 232

infection on LEDGF/p75, and reveal a significant, but secondary role for the CA protein. 233

CA missense mutations or cyclosporine A (CsA) treatment reduces HIV-1 234

dependency on LEDGF/p75. The chimeric viral analysis revealed a role for the CA 235

protein in determining the dependency of HIV-1 infection on LEDGF/p75. Because the 236

HIV-based MHIV-mMA12CA chimera contained the heterologous MLV CA protein, we 237

next analyzed missense mutant viruses to test whether minor changes in CA would alter 238

HIV-1 dependency on LEDGF/p75. Eight CA mutations were selected based on their 239

effects on the dependency of HIV-1 infection on host factors cyclophilin A (CypA), 240

TNPO3, NUP358, or NUP153. The G89V and P90A mutations are within the CypA 241

binding loop of the CA N-terminal domain and reduce CA binding to CypA (5, 65). 242

These mutations also affect the dependency of the virus on TNPO3 (14, 48), NUP358 243

(48), or NUP153 (40). CsA is a small molecule that binds the CypA active site (25), and 244

the A92E and G94D mutations confer CsA-dependence on HIV-1 replication in certain 245

cell types (1, 4); the mutations marginally reduce TNPO3 dependency (14) and cause a 246

modest increase in the dependency of infection on NUP153 (40). Three of the other 247

mutant viruses have been shown to affect the stability of the assembled capsid core. 248

The E45A mutation has a stabilizing effect and T54A/N57A and Q63A/Q67A have 249

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destabilizing effects on the capsid core (21, 64). The infectivities of each of these 250

mutants displayed less dependence on TNPO3 (14, 32, 66) or NUP153 (32, 40) than 251

the WT virus. The final CA mutant, N74D, which was selected because it conferred 252

resistance to the cytoplasmic restriction factor CPSF6-358 (32), reduced the 253

dependency of HIV-1 infection on TNPO3 (14, 32, 48, 66), NUP358 (32, 48), and 254

NUP153 (32, 40, 48). MEF cells were challenged with equal RT-cpm of WT and mutant 255

viral constructs that express luciferase. Though Thys et al. have indicated that 256

pseudotyping with VSV-G significantly reduced the dependency of the N74D mutant on 257

TNPO3 (53), subsequent reports showed that TNPO3 knockdown affected CA mutants 258

carrying VSV-G or HIV-1 envelope glycoproteins similarly (14, 40, 48, 66). The IN active 259

site mutant virus D64N/D116N (N/N) was included as a control because it provides a 260

measure of viral titer in the absence of functional IN activity due to weak expression 261

from unintegrated viral DNA and/or low levels of host-mediated DNA integration (40, 262

43). 263

Previously published reports showed that the Psip1 knockout significantly 264

decreased lentiviral DNA integration (39, 51); however, the relative titer of the N/N 265

mutant virus in the E2(-/-), E1(f/+) MEF cell pair was unaffected (Fig. 2B, black bar) 266

because the absolute titer of this mutant virus (~0.4% of WT; Fig 2A, black bar) is 267

independent of IN-mediated DNA recombination. The reduction in the relative titer of the 268

N/N mutant virus in the E6(-/-), E6(f/f) cell pair (Fig. 2D, black bar) is consistent with a 269

transcriptional defect that we have identified for the HIV-1 long terminal repeat (LTR) in 270

the E6(-/-) cell line and in a subset of other Psip1 knockout MEFs (Y.K and A.E., 271

manuscript in preparation). Five of the tested CA mutants, including Q63A/Q67A, N74D, 272

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G89V, P90A, and A92E, were less affected by the Psip1 knockout (~2 to 2.5-fold) than 273

was the WT virus (Fig. 2B and 2D, compare the black bars). Although the absolute titers 274

of some of the CA mutants, including Q63A/Q67A and G89V, were reduced significantly 275

compared to the WT (Fig. 2A and 2C, black bars), the dependence of the virus on 276

LEDGF/p75 did not correlate with inherent infectivity: T54A/T57A, which was as 277

dependent on LEDGF/p75 as the WT virus (Fig. 2B and 2D), had the lowest absolute 278

titer of any of the CA mutants that we tested (~1% of WT) whereas N74D, which was 279

the least dependent on LEDGF/p75, had the greatest absolute titer (~82% of WT; Fig. 280

2A and 2C, black bars). 281

Either including CsA in the cell culture medium, which disrupts the CypA-CA 282

interaction, or using viruses that have G89V or P90A mutations in CA, increased 283

integration of HIV-1 DNA into gene dense regions of chromosomes (48). The addition of 284

CsA reduced the dependence of the WT virus on LEDGF/p75 by ~2 to 3.5-fold in the 285

knockout cells (Fig. 2A and 2C, compare white bar to light grey bar). CsA also reduced 286

the dependence of many of the CA mutant viruses on LEDGF/p75 (statistical 287

significance in relative titers is denoted by asterisks above brackets in Fig. 2B and 2D). 288

These data suggest that either pharmacological or genetic disruption of the CypA-CA 289

interaction can significantly reduce the dependency of HIV-1 infection on LEDGF/p75. 290

However, this relative increase in infectivity in the presence of CsA, or the 291

corresponding increases in relative titers seen with the Q63A/Q67A or N74D mutants, 292

failed to restore the infectivity to the level seen with the WT virus in the presence of 293

LEDGF/p75. In contrast, the N74D mutation could essentially completely overcome the 294

requirement for TNPO3 (14, 32, 48, 66), NUP153 (32, 40), or NUP358 (48), host factors 295

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implicated in nuclear import that are predictably encountered before the PIC engages 296

chromatin-associated LEDGF/p75. 297

CA mutations alter HIV-1 DNA integration. The presence or absence of 298

LEDGF/p75 influences the efficiency of HIV-1 DNA integration, as well as the 299

distribution of the residual proviruses with respect to bodies and promoter regions of 300

active genes (18, 47). Prior studies of the effects of CA mutations have focused on the 301

distribution of integration sites (33, 48). To determine if CA mutations might also 302

influence the efficiency of integration, the reverse transcription and integration profiles of 303

two relatively LEDGF/p75-independent CA mutant viruses, MHIV-mMA12CA and N74D 304

(Fig. 1 and 2), were assessed by Q-PCR. Because the N74D mutation and the 305

mMA12CA chimera were constructed in two different HIV-1 backgrounds (HIV-1NL4-3 for 306

N74D; HIV-1LAI for mMA12CA), each WT strain was used as a positive control. Due to 307

the transcriptional defect in the HIV-1 LTR identified in E6(-/-) knockout cells, the E2(-/-), 308

E1(f/+) cell pair was used to simplify the interpretation of the results. PCR primers and 309

probes were designed to detect the total amount of HIV-1 DNA [measured at 7 hpi, the 310

approximate peak of reverse transcription; ref. (29)], and the number of integrated 311

proviruses (at 48 h). The alu element, which is the chromosomal sequence routinely 312

used in Q-PCR assays to measure HIV-1 integration in human cells, is primate-specific 313

(15). In the BBL-PCR assay the mouse repeat elements B1, B2, and LINE1 are used to 314

measure the number of proviruses (51). Viral stocks were treated with DNase before the 315

target cells were infected to reduce plasmid carryover from transfected cells. DNAs 316

prepared from cells infected in the presence of 25 µM AZT were analyzed in parallel, 317

and this background was subtracted from the experimental datasets. Absolute levels of 318

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DNA synthesis and integration (Fig. 3A and 3C) were re-graphed as relative levels to 319

assess the dependency of DNA formation on LEDGF/p75 (Fig. 3B and 3D). 320

Consistent with our prior report (51), the total amount of HIV-1 DNA in infected 321

cells at 7 hpi was not significantly affected by the LEDGF/p75 knockout (Fig. 3A). The 322

amount of viral DNA synthesized by the N74D mutant was reduced about two-fold from 323

the WT viral vector independent of target cell type; however, the amount of DNA 324

synthesized by the MHIV-mMA12CA chimera was reduced by about two-fold in the 325

knockout cells (Fig. 3A and 3B). The level of N74D mutant DNA integration in WT cells 326

mirrored the level of reverse transcription (Fig. 3C). Because the level of N74D 327

integration in the knockout cells was similar to the level of the WT virus in these cells, 328

the CA mutant virus revealed about a two-fold increase in the efficiency of viral DNA 329

integration in the knockout cells relative to the WT virus (Fig. 3C and 3D). Comparing 330

the absolute values of MHIV-mMA12CA DNA integration to its matched WT virus in the 331

two cell types (Fig. 3C) revealed a similar two-fold increase in the efficiency of mutant 332

viral DNA integration in the knockout cells (Fig. 3D). The fractional increase in 333

integration frequency moreover mirrored the increase in the relative titer of the N74D 334

mutant virus (compare Fig. 3D to Fig. 3F). However, the impact of the MHIV-mMA12CA 335

mutation on the relative infectious titer was approximately 2.4-fold greater than the 336

impact on provirus formation (Fig. 3D-F). From this we inferred that the impact of the 337

N74D mutation on the overall integration frequency determined, in large part, the impact 338

of the mutation on relative infectious titer. The difference seen between the effects on 339

relative titer and integration frequency of MHIV-mMA12CA could be due to the low level 340

of integration of the parental HIV-1LAI control in the knockout cells, which was less than 341

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two-fold above the assay background, or to some other property of this mutant virus, for 342

example its dependence on cell division for infection (46, 62). 343

Differential effects of the N74D CA mutation and NUP153 and LEDGF/p75 344

depletion on integration site distribution. As discussed above, HIV-1 CA missense 345

mutations, including N74D, reduce the dependency of HIV-1 replication and integration 346

on cellular factors implicated in PIC nuclear import, including TNPO3, NUP358, and 347

NUP153 (14, 32, 40, 48, 66). The distribution of HIV-1 integration sites was similarly 348

affected by TNPO3 or NUP358 depletion (46) or the N74D mutation (48). We and 349

others previously reported that knockdown of NUP153 reduced HIV-1 infection and 350

integration (30, 40), but the effect of depleting this factor on the distribution of the 351

integration sites has not been reported. Integration site libraries were prepared from 352

NUP153 depleted and control cells, which were infected with WT HIV-1 or an isogenic 353

N74D mutant virus (Fig. 4A). HeLa cells were used for this analysis to simplify a 354

comparison of our results and other studies (33, 46, 48). As expected, siRNA-mediated 355

knockdown of NUP153 reduced the titer of the WT virus approximately 10-fold without 356

appreciably affecting the efficiency of infection by the mutant virus (40). Genomic DNA 357

harvested 2 days post-infection was sheared by sonication, repaired, and amplified by 358

ligation-mediated PCR for analysis using Illumina deep sequencing. Resulting 359

sequences from each infection condition were parsed to remove duplicate insertion 360

sites, yielding from approximately 1,300 (WT virus in NUP153 knockdown cells) to 361

23,000 (N74D virus in siControl transfected cells) unique sites for the four infection 362

conditions. The sites were analyzed for features such as genes, gene density, start 363

sites, and transcriptional activity, and were compared among the four sets and to a 364

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computer-generated set of 10,000 random sites (Table 1; see Fig. 5 for statistical 365

analyses). 366

Consistent with a recent report (48), the N74D mutation markedly reduced the 367

tendency of HIV-1 DNA to integrate into gene-dense, transcriptionally active regions of 368

chromosomes; the integration sites for this mutant had an average value of 5.7 369

genes/Mb, which is significantly less than the random calculated value of 7.7 (Table 1 370

and Fig. 5A). The N74D mutation also significantly affected the distribution of integration 371

sites with respect to the other evaluated markers, including within genes, near CpG 372

islands or promoters, and transcriptional activity (Table 1; Fig. 4B and 5A). We note that 373

Schaller et al. (48) previously reported similar significant responses for the N74D mutant 374

virus, with the exception of integration near CpG islands. We conjecture that these 375

effects of the N74D mutation on DNA integration site selection may be a contributing 376

factor to the reduced replication capacity of this mutant that can be observed under 377

conditions of spreading viral replication (2, 48). 378

Depletion of NUP153 from target cells also caused statistically significant 379

reductions in the tendencies of viral DNA to integrate into gene dense, transcriptionally 380

active regions of chromosomes, though these effects were less dramatic than the 381

effects caused by the N74D mutation (Table 1, Fig. 4B and Fig. 5A). Although it is 382

somewhat difficult to compare results across studies, the reduction in the tendency of 383

HIV-1 to target gene dense regions upon NUP153 depletion would appear to be more 384

modest than the magnitude of the reductions caused by either TNPO3 or NUP358 385

depletion (46). NUP153 depletion did not significantly influence the distribution of WT 386

HIV-1 integration sites with respect to any of the other evaluated markers (Table 1 and 387

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Fig. 5A). Because the N74D mutant virus infects control and NUP153 knockdown cells 388

with similar efficiencies (Fig. 4A), the distribution of the integration sites of the mutant 389

virus in cells depleted for NUP153 predictably mimicked the proviral distributions 390

observed with siControl-treated cells (Table 1, Fig. 4B and 5A). We note that neither 391

NUP153 depletion nor the N74D mutation affected the local DNA sequence specificity 392

at the site of HIV-1 integration (data not shown). 393

Although prior reports failed to ascribe a role for LEDGF/p75 in the targeting of 394

HIV-1 integration to gene dense regions of chromosomes (39, 46), we found that 395

mutations in CA can significantly alter the dependency of virus infection on the 396

integration co-factor (Fig. 1 and 2), prompting us to re-evaluate the role of LEDGF/p75 397

in this process. Integration site libraries were prepared from matched LEDGF/p75-398

expressing E1(f/+) and E2(-/-) knockout cells infected with either the WT or N74D CA 399

mutant virus, which yielded from approximately 92,500 (WT infection of Psip1 knockout 400

cells) to 524,000 (N74D virus in WT cells) unique sequences among the four infection 401

conditions (Table 2). As expected (39, 49, 51), the integration of the WT virus within 402

genes was reduced significantly, from 73.5% to 49.4%, by the knockout, with 403

concomitant increases in targeting to CpG islands and gene promoter regions (Table 2). 404

The integration distribution of the N74D mutant virus was also significantly affected by 405

the knockout, with integration into genes dropping from 59.0% in the presence of 406

LEDGF/p75 to 39.0% in its absence (Table 2). Thus, although the efficiency of N74D 407

mutant virus integration is less affected by the Psip1 knockout than is the efficiency of 408

WT virus integration, the distribution of mutant viral DNA integration, in large part, 409

remains LEDGF/p75-dependent (Fig. 3C and 3D). Accordingly, N74D, like the WT virus, 410

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favors gene promoter regions and CpG islands in the absence of LEDGF/p75, even 411

though the mutant virus tends to avoid these elements in the presence of LEDGF/p75 412

(Table 1 and 2; Fig. 5). As observed in HeLa cells, the N74D mutation significantly 413

reduced the tendency of the PIC to target active genes (Fig. 4B and 4C) and greatly 414

reduced integration into transcriptionally active, gene dense regions of mouse 415

chromosomes (Table 1 and 2; Fig. 5). The Psip1 knockout moreover reduced the 416

average gene density for integration of the WT virus, from 18.2 to 14.8 genes/Mb, a 417

statistically significant difference (Table 2 and Fig. 5B). We suggest that the difference 418

in the significance of targeting of transcriptionally active, gene-enriched regions of 419

chromosomes observed here compared to previous reports (39, 46) may be due to 420

different genomic annotations [Marshall et al. (39) utilized mouse genome build mm8, 421

whereas mm9 was utilized here] and/or the deeper coverage in our integration site 422

libraries. 423

Conclusions. This study extends our knowledge of the effects that CA mutations 424

and host cell factors have on the efficiency of HIV-1 integration and distribution of the 425

resulting proviruses. In addition to affecting the targeting of gene dense regions of 426

chromosomes (48), we show that CsA treatment and CA mutations reduced the 427

dependency of the virus on LEDGF/p75 (Fig. 1 and 2), which correlates with their 428

effects on integration efficiency (Fig. 3). The N74D mutation reduced integration into the 429

bodies of active genes and eliminated the targeting of transcriptionally active, gene-430

dense regions of chromosomes, whereas NUP153 played a subsidiary role in the 431

targeting of gene dense regions (Table 1 and 2; Fig. 4 and 5). By analyzing relatively 432

large datasets, we show that chromatin-associated LEDGF/p75 also plays a role in the 433

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targeting of HIV-1 integration into gene-dense regions of chromosomes (Table 2 and 434

Fig. 5B). It would appear that LEDGF/p75 plays the dominant role in targeting 435

integration into the bodies of active genes, whereas host factors associated with PIC 436

nuclear import play the predominant role in the targeting of gene dense regions of 437

chromosomes (46). Analysis of cells depleted for nuclear transport factors by gene 438

knockout, which have been described for NUP358 (13), would help to clarify potential 439

contributions of low levels of nucleo-cytoplasmic transport proteins to HIV-1 infection 440

and viral DNA integration. 441

The relative insensitivity of HIV-1 CA mutant viruses to LEDGF/p75 ablation 442

supports at least two potential models. Based on the relatively large gain in infectious 443

titer by the mMA12CA chimeric virus, we suggest that the HIV-1 CA protein, either 444

indirectly or through a direct interaction with IN, impacts the functional interaction 445

between LEDGF/p75 and the viral recombinase. Concordantly, relatively minor CA 446

changes, like the N74D missense mutation, in large part retain the IN-LEDGF/p75 447

interaction. Our data also raise the alternative possibility that HIV-1 CA might interact 448

with a cellular factor that bridges it to LEDGF/p75. Given the similarity between the 449

effects of a TNPO3-knockdown and the N74D mutation on HIV-1 integration site 450

selectivity (46, 48), a potential role for TNPO3 or TNPO3-interacting factors should be 451

considered. Notably, it has also been reported that HIV-1 CA can enter the nucleus 452

during infection, but that the transport of the N74D mutant CA into the nucleus is 453

diminished relative to the WT virus (66). Whether the mode of trafficking through the 454

NPC, or the presence of CA in the PIC, or both influences the subsequent interactions 455

of IN with nuclear factors, including LEDGF/p75, will be important to determine. 456

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An obvious limitation of our approach is the possibility that knockdown or 457

knockout of the cell factors could have an indirect effect on the distribution of the 458

integrated proviruses. NUP-associated regions of chromatin, which encompass 459

approximately 25% of the Drosophila genome (58), are enriched for markers of active 460

transcription, including RNA polymerase II occupancy and histone H4K16 acetylation. It 461

therefore seems likely that depletion of NPC components could significantly affect 462

chromatin organization and/or the distribution of the chromosomes within the nucleus, 463

which could, in turn, affect the efficiency and/or distribution of viral DNA integration. 464

Although LEDGF/p75 is a constitutive chromatin component and its depletion influences 465

the expression of large numbers of cellular genes (12), the fact that novel LEDGF/p75 466

fusion proteins that contain foreign chromatin binding domains quantitatively redirect 467

HIV-1 integration (20, 23, 52), shows that LEDGF/p75 has a direct role in HIV-1 DNA 468

integration targeting. 469

Fortunately, the question of whether a host gene knockdown can indirectly 470

influence integration site selection does not apply to the data obtained with the N74D 471

mutant virus. Biochemical studies that characterize the details of virus-host interactions 472

can also help to delineate direct versus indirect effects. Two different NUPs, NUP153 473

(59) and NUP62 (3), have been shown to bind to HIV-1 IN, suggesting it may be 474

possible to configure novel targeting constructs based on these factors as well. Our 475

finding that mutations in CA, which is the key HIV-1 determinant of PIC nuclear import 476

(62, 64), alter the dependency of the virus on cellular LEDGF/p75 supports a model 477

whereby the mechanism of PIC trafficking is linked to the efficiency and specificity of 478

viral DNA integration. 479

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480

ACKNOWLEDGEMENTS 481

The authors thank Charles Bangham for sharing the method of integration site 482

library construction prior to publication, Greg Towers for stimulating discussion in the 483

early phase of this project, and Masahiro Yamashita and Michael Emerman for their 484

generous gift of parental and MHIV chimera reporter constructs. 485

This work was supported by the National Cancer Institute’s Intramural Center for 486

Cancer Research, which supports the HIV Drug Resistance Program (V.N.K., S.H.H.), 487

as well as National Institutes of Health (NIH) grants AI039394 and AI052014 (A.E.). 488

The contents of this article or mention of commercial reagents is in no way meant 489

to reflect the options of, or endorsement by, the NIH or U.S. Government. 490

491

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58. Vaquerizas, J. M., R. Suyama, J. Kind, K. Miura, N. M. Luscombe, and A. Akhtar. 677 2010. Nuclear pore proteins nup153 and megator define transcriptionally active regions 678 in the Drosophila genome. PLoS Genet. 6:e1000846. 679

59. Woodward, C. L., S. Prakobwanakit, S. Mosessian, and S. A. Chow. 2009. Integrase 680 interacts with nucleoporin NUP153 to mediate the nuclear import of human 681 immunodeficiency virus type 1. J. Virol. 83:6522-6533. 682

60. Wu, X., Y. Li, B. Crise, and S. M. Burgess. 2003. Transcription start regions in the 683 human genome are favored targets for MLV integration. Science 300:1749-1751. 684

61. Wu, X., Y. Li, B. Crise, S. M. Burgess, and D. J. Munroe. 2005. Weak palindromic 685 consensus sequences are a common feature found at the integration target sites of 686 many retroviruses. J. Virol. 79:5211-5214. 687

62. Yamashita, M., and M. Emerman. 2004. Capsid is a dominant determinant of retrovirus 688 infectivity in nondividing cells. J. Virol. 78:5670-5678. 689

63. Yamashita, M., and M. Emerman. 2005. The cell cycle independence of HIV infections 690 is not determined by known karyophilic viral elements. PLoS Pathog. 1:e18. 691

64. Yamashita, M., O. Perez, T. J. Hope, and M. Emerman. 2007. Evidence for direct 692 involvement of the capsid protein in HIV infection of nondividing cells. PLoS Pathog. 693 3:1502-1510. 694

65. Yoo, S., D. G. Myszka, C. Yeh, M. McMurray, C. P. Hill, and W. I. Sundquist. 1997. 695 Molecular recognition in the HIV-1 capsid/cyclophilin A complex. J. Mol. Biol. 269:780-696 795. 697

66. Zhou, L., E. Sokolskaja, C. Jolly, W. James, S. A. Cowley, and A. Fassati. 2011. 698 Transportin 3 promotes a nuclear maturation step required for efficient HIV-1 integration. 699 PLoS Pathog. 7:e1002194. 700

701 702 Figure legends 703

FIG 1 Both CA and IN affect the dependence of HIV-1 infection on LEDGF/p75. 704

(A) Schematic drawing of the parental HIV-1 (white boxes) and MLV (gray boxes) Gag 705

and Pol proteins, and the chimeric viruses. PR, protease (other abbreviations defined in 706

the text). Note that the MLV Gag p12 protein does not have a corresponding 707

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counterpart in HIV-1. (B) Absolute viral titers [left graph; black, E1(f/+) cells; white, E2(-/-708

) cells] and relative viral titers of HIV-1, MLV, and MHIV chimerae in E2(-/-) knockout 709

cells expressed as a percentage of the E1(f/+) control cell infections (right graph) are 710

shown. Data are mean ± standard error of mean (SEM) for two independent 711

experiments, each conducted in duplicate. Right, statistical comparison of relative titer 712

versus WT HIV-1 (notation above bar) or between chimera viruses (brackets) was 713

determined by paired two-tailed Student’s t-test: n.s, not significant (P > 0.05); *, P < 714

0.05; **, P < 0.005. (C) Same as in panel B, except that the E6(f/f) WT and E6(-/-) 715

knockout MEF cell pair was analyzed. In this case, the MHIV-mMA12CA to WT 716

comparison yielded a P value of 0.052. 717

718

FIG 2 Infectivity of CA missense mutants in the presence and absence of 719

LEDGF/p75. (A) Absolute titers of WT and indicated CA mutant viruses in E1(f/+) WT 720

and E2(-/-) cells. The black and dark gray bars are viral infectivities in E1(f/+) cells 721

minus versus plus CsA, respectively, expressed as percent WT virus infection in the 722

absence of CsA, which was set at 100%. Light gray and white bars, infectivities in E2(/-) 723

cells without versus with CsA, respectively. (B) Relative titers of WT and CA mutant 724

viruses in knockout E2(-/-) cells are expressed as a percentage of their infectivity in 725

control E1(f/+) cells. White bars, infections conducted in the presence of CsA. Results 726

are averages of three to five experiments performed in duplicate, with error bars 727

denoting 95% confidence intervals. Asterisks directly above the bars reveal statistical 728

relevance in comparison to the WT virus (determined by paired two-tailed Student’s t-729

test; *, P < 0.05; **, P < 0.005) without versus with CsA; asterisks above brackets 730

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indicate relevant titer differences for the indicated viruses +/- CsA. (C and D) Same as 731

in panels A and B, except that the E6(f/f) and E6(-/-) MEF cell pair was analyzed in 732

three independent experiments. 733

734

FIG 3 HIV-1 DNA synthesis and integration in the presence and absence of 735

LEDGF/p75. (A) Total HIV-1 DNA levels at 7 hpi, expressed as percentage of the 736

indicated WT virus in E1(f/+) cells. (B) Panel A data were re-graphed to highlight the 737

DNA levels observed in the knockout cells relative to control LEDGF/75-expressing 738

cells. The dotted line represents background values obtained from corresponding AZT-739

treated samples, which averaged 1.8% of the level seen in untreated cells. (C and D) 740

Same as in panels A and B, except that the BBL-PCR assay measures the number of 741

integrated proviruses at 48 hpi; the AZT plus background in panel D was 7.7% of the 742

uninhibited control. (E) Absolute virus infectivities determined by processing fractions of 743

panel A-infected cells for luciferase activity and protein content at 48 hpi. (F) Relative 744

titers of the viruses in E2(-/-) versus control E1(f/+) cells. Panels A-D show averages 745

and standard errors from duplicate Q-PCR assays that are representative of two 746

independent experiments. Panels E and F average the results of two experiments, with 747

the error bars denoting 95% confidence intervals. Although comparisons of relative 748

amounts of mutant viral DNA (panel D) and titers (panel F) to the matched WT controls 749

failed to achieve statistical significance in these experiments, we note that the fractional 750

increase in N74D mutant viral DNA integration versus the WT virus, 2.1-fold (panel D), 751

virtually matched the fractional difference in N74D-to-WT viral infectivity (1.9-fold, panel 752

F). 753

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754

FIG 4 Infections and integration site preferences as a function of gene 755

expression. (A) HeLa cells transfected with siNUP153#1 (light gray bars) or the siRNA 756

control siControl (dark grey bars) (40) were infected with the indicated virus. The 757

reported titers were calculated based on WT virus infection of siControl-transfected 758

cells, which was set to 100%. Data are the mean ± SEM from two independent 759

experiments. NUP153 depletion yielded a significant reduction in WT viral titer (P = 760

0.026) as calculated by a one sample, one-tailed t-test. (B) Integration frequency as a 761

function of gene expression in HeLa cells. The x-axis represents >16,000 well-762

characterized human genes placed into bins of 100 according to level of gene 763

expression. The number of integrations in each bin is plotted for the four different 764

infection conditions (Fig. 4A and Table 1) versus the random calculated average. (C) As 765

in panel B, except that integration as a function of gene expression was analyzed in 766

mouse cells (Table 2). 767

768

FIG 5 Statistical analyses of HIV-1 integration distributions. (A) Fisher’s exact 769

test was utilized to analyze the distributions of integration within genes, and near 770

transcriptional start sites (TSSs) and CpG islands, in the indicated pairwise 771

configurations for the data presented in Table 1. The Mann-Whitney U test (Wilcox rank-772

sum test) was used to analyze the number of average genes/Mb and associated 773

transcriptional activity. P values greater than 0.05 are highlighted in bold, italic type. (B) 774

Mann-Whitney U test results of pairwise analyses of integration into gene dense regions 775

of chromosomes and associated transcriptional activity in MEF cells (Table 2) are 776

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shown. Fisher’s exact test of all pairwise combinations of integration values within 777

genes as well as near promoters and CpG islands yielded P values < 0.0001 (matrices 778

not shown). 779

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TABLE 1 Integration Sites in Control and Knockdown HeLa Cells 781

Integration sitesa 782 WT_siCtr WT_si153 N74D_siCtr N74D_si153 Random 783

Genomic Feature n = 5,390 n = 1,318 n = 22,682 n = 9,610 n = 10,000 784 Transcription unitsb 3,604 (66.9) 902 (68.4) 13,582 (59.9) 5,504 (57.3) 3,960 (39.6) 785 Promoters (± 1 kb) 40 (0.74) 15 (1.14) 111 (0.49) 49 (0.51) 167 (1.67) 786 CpG Island (± 1 kb) 17 (0.32) 2 (0.15) 19 (0.08) 10 (0.10) 81 (0.81) 787 Genes per 1 Mb 12.2 10.9 5.7 5.5 7.7 788 Transcriptional activityc 400 316 120 119 173 789 a Numbers in parentheses are percentage values. 790 b Refseq genes from human genome build hg19, UCSC Genome Bioinformatics website. 791 c Total RNA tag counts per Mb surrounding each integration site. 792

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TABLE 2 Integration Sites in Control and LEDGF/p75 Knockout MEFs 794 Integration sitesa 795

WT_WT N74D_WT WT_Psip1-/- N74D_Psip1-/- Random 796 Genomic Feature n = 273,049 n = 524,372 n = 92,475 n = 440,756 n = 100,000 797 Transcription unitsb,c 200,760 (73.5) 309,381 (59.0) 45,695 (49.4) 171,574 (39.0) 33,031 (33.0) 798 Promoters (± 1 kb)c 1,866 (0.68) 2,636 (0.50) 3,540 (3.83) 10,065 (2.28) 1,595 (1.60) 799 CpG Island (± 1 kb)c 1,428 (0.52) 882 (0.17) 2,422 (2.62) 4,884 (1.11) 728 (0.73) 800 Genes per 1 Mb 18.2 7.7 14.8 8.0 8.7 801 Transcriptional activityd 587 222 464 231 249 802 a Percentage values are indicated in parentheses. 803 b Refseq genes from mouse genome build mm9, UCSC Genome Bioinformatics website. 804 c Pairwise comparisons by Fisher’s exact test in all cases yielded P values less than 0.0001. 805 d Total RNA tag counts per Mb surrounding each integration site. 806

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Differential Effects of Human Immunodeficiency Virus Type 1 Capsid

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Transcript of Differential Effects of Human Immunodeficiency Virus Type 1 Capsid