Vol. 64, No. 5

JOURNAL OF VIROLOGY, May 1990, p. 2421-2425

0022-538X/90/052421-05$02.00/0 Copyright C 1990, American Society for Microbiology

Integration Is Not Necessary for Expression of Human Immunodeficiency Virus Type 1 Protein Products MARIO STEVENSON,* SHERYL HAGGERTY, CAROLYN A. LAMONICA, CRAIG M. MEIER, SIAO-KUN WELCH,t AND ANDRZEJ J. WASIAK

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68105-1065 Received 1 September 1989/Accepted 23 January 1990

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.

function of the pol gene, since constructs containing pol sequences up to position 4153 produced wild-type levels of reverse transcriptase in a procaryotic expression system (6). The 3' terminal 146 amino acids were deleted from integrase mutants HIV-1 Aint2, Aint5, and Aint52. In addition, the deletions in HIV-1 Aint2 and Aint5 resulted in a frameshift to the + 1 reading frame and the substitution of 30 and 18 amino acids, respectively (Fig. 1). The deletion in HIV-1 Aint52 resulted in a frameshift to the -1 reading frame and a substitution of eight amino acids (Fig. 1). Integrase mutant HIV-1 Aint29 contained an in-frame deletion of 52 amino acids. In addition, an insertion mutant (HIV-1 Aintl2p) generated by the introduction of concatemerized EcoRI linkers at map position 4647 resulted in an additional 21 in-frame amino acids compared with the wild-type protein. In all integrase mutants containing frameshifts, the presence of stop codons in the -1 (position 4783 of HXB2) and + 1 (position 4827) frames prevented shifting of the integrase reading frame into that of VIF. Wild-type HIV-1 mfA and integrase mutant virus were rescued following transfection of human HeLa cells and astroglial cells (U251-mg) (2) by DNA-calcium phosphate coprecipitation, resulting in the establishment of an artificially integrated provirus. This did not require a reverse transcriptase step, and integration occurred at any point on the proviral clone. The production of virus antigen from this artificially integrated provirus and the release of virions were monitored by a highly sensitive and specific p24 antigen detection assay (Coulter Immunology, Hialeah, Fla.). Wildtype HIV-1 mfA isolates and deletion mutants HIV-1 Aint2, Aint5, Aint29, and Aint52 produced abundant virus from transfected monolayer cultures (results not shown). Surprisingly, cultures transfected with mutant HIV-1 Aintl2p bearing amino acid insertions within the HIV-1 integrase were repeatedly negative for the presence of p24 in the supematant despite the presence of p24 within the transfected cells, indicating an inability of these mutants to assemble virus components and produce virions. Wild-type HIV-1 mfA virions and integrase mutants were harvested from the supernatants of transfected HeLa and U251-mg cultures

The human immunodeficiency virus type 1 (HIV-1) exerts profound cytopathic effects on host CD4 lymphocytes in vitro (1, 13). The CD4 lymphocyte is the major reservoir for HIV-1 in vivo (17), and the effect of HIV-1-mediated cytolysis on the cell population may account for the CD4 lymphocyte depletion during the progression of acquired immunodeficiency syndrome (5). The molecular basis for HIV1-mediated cytolysis is incompletely characterized, but as with all cases of cytocidal retrovirus infection studied to date, including avian reticuloendotheliosis virus, avian leukosis virus, visna virus, and feline leukemia virus (7, 11, 14, 16, 20, 25, 28), it is characterized by the accumulation of unintegrated viral DNA and unusually high levels of RNA and protein. Similarly, cytocidal HIV-1 infection is characterized by a rapid accumulation of unintegrated viral DNA (20, 23) and the generation of very high levels of viral RNA and protein (22). It has been suggested that the accumulation of unintegrated viral DNA plays a central role in the cytopathogenesis of retroviral infections (28). Studies with Moloney murine leukemia virus indicate that integration is required for a productive infection (18), although integration-defective mutants of spleen necrosis virus were able to produce virus, suggesting that unintegrated viral DNA is a template for virus antigen production (12). The viral determinant required for integration of the provirus is located at the 3' terminus of the viral pol gene. A series of HIV-1 mutants containing deletions in the integrase/endonuclease-coding region of an infectious proviral DNA clone (HIV-1 mfA) were constructed as outlined in Fig. 1. The coordinates of the HIV-1 integrase have been assigned through direct sequencing of its amino terminus (9). The presence of an EcoRI restriction site 419 bases downstream of the amino terminus of the HIV-1 integrase (map position 4647 of HIV isolate HXB2) (15) facilitated the construction of mutants with extensive deletions in the integrase gene. Such deletions (from map position 4647) would not be expected to affect the reverse transcriptase *

Corresponding author.

t Present address: Schering Health, Omaha, NE 68103. 2421

2422

NOTES

J. VIROL. LTR -

r

ij

GAG

vpI

POL

B

CLONE

FRAMESHIFr

INTEGRASE LENGTH

K K

ES

E

ENV

K

nof

AMINO ACID SEQUENCE

137

HIV-I NrfA

LTR --

:

f

165

mi- i Al ..ii

I

289

OEFGIP YNPOSOGWESMNKELKKIIGQV -_

rvI iff" IIt I

(,-n If FPIif

(Wild Type)

I

i

6

ir

HL i 41 A 61

HIV-I Aint 52

-1

150

QEFGIP PGSIHPQF*

HIV-I A int 2

+I

173

OEFGIP NILRQQYKWQYSSTILKEKGGLGGTVQGKE*

HIV-I A int 5

+I

161

QEFGIP STILKEKGGLGGTVQGKE*

I

mi- a A I

iry"

.Ii

im

L

mi- i J..j T

.1

'FF"

I

mi- i

p195

HIV-I A int 29

In Frame

237

QEFGIP SAGERIVDIIATDIOT -_-

HIV-I A intl2p

In Frame

309

OEFG LIFRNSGIPEFRNSGIPEFQF IPYNPOS_

I

-1

Wmin"pry

T

L. I

r-f

FIG. 1. Mutant HIV-1 clones containing integrase deletions and insertions. Mutations in the integrase-coding region were generated in full-length infectious proviral DNA clone HIV-1 mfA (M. Stevenson, C. Lamonica, C. Meier, V. Vlach, A. M. Mann, C. Borgeson, and A. Wasiak, submitted for publication). The amino terminus of the HIV-1 integrase, assigned by direct amino acid sequencing (9), begins with a phenylalanine (F) residue (map position 4228 of HIV-1 HXB2) (15). The integrase gene terminates at position 5093, resulting in a protein of 289 amino acids (34 kilodaltons). A 1,095-base-pair EcoRI fragment of HIV-1 mfA containing the 3' half of the HIV-1 integrase gene was subcloned in the EcoRI site of pUC18. Deletions originating from the 5' EcoRI site were generated by digestion with BAL 31, blunt ended with T4 DNA polymerase, ligated to EcoRI linkers, and reinserted at the EcoRI site of HIV-1 mfA. The predicted amino acid sequence of each mutant integrase product was determined from the DNA sequence of each mutant. Insertion mutants were generated by partial EcoRI digestion, blunt end ligated to EcoRI linkers, and recircularized following transformation of Escherichia coli. Numbered residues indicate the locations of wild-type amino acids relative to the first codon of the HIV-1 integrase protein. Where frameshift deletions have been introduced, substituted amino acids are indicated in one-letter amino acid code, while arrowheads identify the junction between wild-type and substituted sequences. Predicted hydrophobicity plots of complete wild-type and mutant integrase proteins are indicated, with hydrophilic residues (above the line) positive. Arrows indicate the junctions between wild-type and mutated amino acid sequences. Restriction endonucleases shown below the genome map of HIV-1 are B, BglII; K, KpnI; E, EcoRI; and S, Sall. *, Termination codon.

after 48 h and allowed to infect CD4+ cells, including the human T-cell leukemia virus type 1- and type 2-positive T-cell lines MT-4 (10) and Mo-T (3) and primary phytohemagglutinin-stimulated cord blood lymphocytes. Transient transfection of HeLa cells with mutant and wild-type DNA clones generated low-titer virus stocks which, upon infection of MT-4 or Mo-T cells, resulted in less than 0.1% HIV-1 antigen-positive cells by 48 h postinfection (Table 1). Serum used for the immunofluorescence assay had a high envelope titer, indicating that unintegrated templates were competent for HIV-1 envelope glycoprotein production. In addition, in all three cell types used in this study (peripheral blood

lymphocytes, Mo-T, and MT-4), infection with wild-type HIV-1 mfA virions and integrase mutants resulted in the synthesis of HIV-1 p24 antigen, as determined by a gag p24 enzyme-linked immunosorbent assay (Table 1), although integrase mutant HIV-1 Aint52 produced barely detectable levels of p24 in infected Mo-T and MT-4 cultures. The variability in p24 production by cells infected with wild-type and mutant viruses appeared to be reflected primarily by the number of infected cells in the culture following virus exposure (results not shown). Supernatant from HeLa cultures transfected with insertion mutant HIV-1 Aintl2p provided a mock-infected control, since this clone repeatedly

NOTES

VOL. 64, 1990

failed to produce virus following transfection of HeLa or U237-mg cells. The p24 antigen production observed within infected cells was not due to the detection of residual input virus, since in all cases the level of p24 production within infected cells was at least 10 times higher than the amount of p24 in the virus inoculum (Table 1). Culture supernatants of Mo-T and MT-4 cells infected with wild-type virions and each of the deletion mutants contained abundant HIV-1 p24 antigen, although in MT-4 cells there was a reduced level of p24 antigen from cultures infected with the integrase deletion mutants. Electron microscopic analysis of integrase-minus HIV-1-infected Mo-T cells failed to demonstrate the presence of virus particles, although the low level of infection in this system made such detection difficult. In addition, analysis of infected Mo-T cultures 8 and 15 days postinfection did not reveal the presence of any HIV-1 antigen-positive cells in cultures infected with integrase mutants (as evidenced by immunofluorescence analysis), while antigen-positive cells were present in wild-type HIV-1 mfA-infected cultures (Table 1). Similarly, it was not possible to establish infection of Mo-T or MT-4 cells following inoculation with supernatants from Mo-T cells containing proviral integrase mutant genomes. This indicated that either deletion mutants were unable to set up a productive infection in other cells or the kinetics of replication and infection of the deletion mutants were extremely low when compared with those of wild-type virus. To ensure that the observed HIV-1 p24 antigen production within cells infected with integration mutants originated from unintegrated viral DNA, we used the polymerase chain reaction technique to search for viral DNA within the genomic fraction and the extrachromosomal fraction (Hirt fraction) of infected cells. One set of primers complementary

TABLE 1. p24 antigen production in CD4+ cells infected with HIV-1 integrase mutantsa HIV-1 p24 antigen production'

Cord

Clone

HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1

MT-4

blood (cell associated)

Cell associated

Supernatant

769 ND 487 650 615