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PubMed1998

Regulation of alphavirus 26S mRNA transcription by replicase component nsP2.

Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII+RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 degrees C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15,000 g (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.

Alphavirus↗

PubMed1999

Diversity of human endogenous retrovirus class II-like sequences.

Class II human endogenous retroviruses (HERVs), often referred to as mouse mammary tumour virus (MMTV)-like or HERV-K elements, have similarities to several animal infectious retroviruses. Single clones from each of nine class II HERV groups (NMWV 1 to NMWV 9), isolated from a human breast cancer cell genomic library, were sequenced over a 244 bp stretch of the conserved reverse transcriptase region. These sequences were aligned to related exogenous and endogenous retroviruses and a phylogenetic tree was constructed. Sequences with more than 80% identity were considered as members of one group and we report here that the class II HERV family consists of at least ten groups. Three of the sequenced clones, from groups NMWV 3, 7 and 9, could not be related to any other previously identified elements and constituted their own groups. NMWV 8 had no similarity to any retroviral sequences in the sequenced region and is so far considered to be non-retroviral.

Amino Acid Sequence↗

PubMed1998

Requirement of cauliflower mosaic virus open reading frame VI product for viral gene expression and multiplication in turnip protoplasts.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

Blotting, Northern↗

PubMed2001

Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Animals↗

PubMed2006

Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication.

A recent report sought to demonstrate that acetylation of specific lysines within integrase (IN) by the histone acetyltransferase (HAT) p300 regulates human immunodeficiency virus type 1 (HIV-1) integration and is essential for viral replication (A. Cereseto, L. Manganaro, M. I. Gutierrez, M. Terreni, A. Fittipaldi, M. Lusic, A. Marcello, and M. Giacca, EMBO J. 24:3070-3081, 2005). We can corroborate the efficient and specific acetylation of the IN carboxyl-terminal domain (CTD) (amino acids 212 to 288) by p300 using purified recombinant components. Although arginine substitution mutagenesis of the isolated CTD confirms that the majority of p300 acetylation occurs at lysine residues 264, 266, and 273, the pattern of acetylation is not uniform and a hierarchy of reactivity can be established. Several combinatorial mutations of the CTD lysines modified by p300 in vitro were reconstructed into an otherwise infectious proviral plasmid clone and examined for viral growth and frequency of productive chromosomal integration. In contrast to the findings of Cereseto and coworkers, who used epitope-tagged viruses for their experiments, we find that an untagged mutant virus, IN K(264/266/273)R, is fully replication competent. This discrepancy may be explained by the use of an acidic epitope tag placed at the extreme carboxyl terminus of integrase, near the target site for acetylation. Although the tagged, wild-type virus is viable, the combination of this epitope tag with the RRR substitution mutation results in a replication-defective phenotype. Although IN belongs to the very small set of nonhistone proteins modified by HAT-mediated activity, an obligate role for acetylation at the reactive CTD lysines in HIV-1 IN cannot be confirmed.

Acetylation↗

PubMed1988

Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.

Codon↗

PubMed1988

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes.

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.

Animals↗

PubMed1990

Simian immunodeficiency viruses from African green monkeys display unusual genetic diversity.

African green monkeys are asymptomatic carriers of simian immunodeficiency viruses (SIV), commonly called SIVagm. As many as 50% of African green monkeys in the wild may be SIV seropositive. This high seroprevalence rate and the potential for genetic variation of lentiviruses suggested to us that African green monkeys may harbor widely differing genotypes of SIVagm. To investigate this hypothesis, we determined the entire nucleotide sequence of an infectious proviral molecular clone of SIVagm (155-4) and partial sequences (long terminal repeat and Gag) of three other distinct SIVagm isolates (90, gri-1, and ver-1). Comparisons among the SIVagm isolates revealed extreme diversity at the nucleotide and amino acid levels. Long terminal repeat nucleotide sequences varied up to 35% and Gag protein sequences varied up to 30%. The variability among SIVagm isolates exceeded the variability among any other group of primate lentiviruses. Our data suggest that SIVagm has been in the African green monkey population for a long time and may be the oldest primate lentivirus group in existence.

Amino Acids↗

PubMed1991

Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus.

Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus.

Base Sequence↗

PubMed1991

Three poliovirus 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus.

Many functions of the poliovirus genome in virally infected cells have been elucidated. However, the role of 2B (and of its precursor polypeptide, 2BC), encoded by the P2 region in the poliovirus genome, remains unknown. We have employed a genetic approach to examine the role of 2B in poliovirus-infected cells. We report here the phenotype of one previously isolated mutant in the 2B coding region, 2B201. In addition, we have constructed one additional mutation in the 2B coding region of an infectious poliovirus cDNA clone. Upon transfection into monkey Vero cells we could recover two 2B mutant polioviruses, 2B204 and 2B205. All three mutants exhibited small-plaque phenotypes on monkey Vero and human HeLa cells and displayed primary defects in viral RNA synthesis. None of the 2B mutants could be complemented by wild-type virus. Instead, the mutants exhibited a dosage-dependent dominance over wild-type poliovirus. Thus, the phenotypes of these 2B mutants implicate 2B and possibly its precursor, 2BC, in viral RNA amplification in poliovirus-infected cells, and the dominance of the 2B mutants suggests a structural role for 2B in viral replication complexes.

Animals↗

PubMed1992

Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.

Amino Acid Sequence↗

PubMed1992

Mutations in the principal neutralization determinant of human immunodeficiency virus type 1 affect syncytium formation, virus infectivity, growth kinetics, and neutralization.

The principal neutralization determinant (PND) of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains a conserved GPG sequence. The effects of a 29-amino-acid deletion of most of the PND, a 3-amino-acid deletion in the GPG sequence, and 16 single-amino-acid substitutions in the GPG sequence were determined in a transient expression assay. All mutant envelope glycoproteins were expressed at levels comparable to that of the wild-type envelope, and mutations in the GPG sequence did not affect processing to gp120 or, except for the 29-amino-acid deletion, binding to CD4. Of all of the mutants, only the GHG and GFG mutants induced formation of syncytia similar in size and number to those induced by the wild-type envelope. When the envelope expression level was increased 10-fold or more, several additional mutants (APG, GAG, GSG, GQG, GVG, and GPF) also induced syncytium formation. Transfection with infectious proviral molecular clones containing the GHG, GFG, APG, GAG, GSG, or GPF mutations induced production of viral particles; however, only the GPG, GHG, and GFG viruses produced active infections in CD4-bearing cells. Furthermore, whereas the wild-type virus was efficiently neutralized by PND polyclonal and monoclonal antibodies, the GHG- and GFG-containing viruses were not. These results show that mutations in the GPG sequence found within the PND do not affect envelope expression and do not significantly affect CD4 binding or production of viral particles but that they do affect the ability of the envelope to induce syncytia and those of the viral particles to infect CD4 cells and be neutralized by PND antibodies.

Amino Acid Sequence↗

PubMed1993

Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus.

The importance of the vpr gene for simian immunodeficiency virus (SIV) replication, persistence, and disease progression was examined by using the infectious pathogenic molecular clone called SIVmac239. The ATG start codon of the vpr gene was converted to TTG by site-specific mutagenesis. The constructed Vpr- mutant virus is identical with the parental SIVmac239/nef-stop virus with the exception of this one nucleotide. These viruses replicated with similar kinetics and to similar extents in rhesus monkey lymphocyte cultures and in the human CEMX174 cell line. Five rhesus monkeys were inoculated with the Vpr- variant of SIVmac239/nef-stop, and two monkeys received SIVmac239/nef-stop as controls. Both controls showed reversion of the TAA stop signal in nef by 2 weeks postinfection, as has been observed previously. Reversion of the TAA stop codon in nef also occurred in the five monkeys that received the Vpr- variant, but reversion was delayed on average to about 4 weeks. Thus, the mutation in vpr appeared to delay the rapidity with which reversion occurred in the nef gene. Reversion of the TTG sequence in vpr to ATG was observed in three of the five test animals. Reversion in vpr was first observed in these three animals 4 to 8 weeks postinfection. No vpr revertants were found over the entire 66 weeks of observation in the other two test animals that received the vpr mutant. Antibodies to vpr developed in those three animals in which reversion of vpr was documented, but antibodies to vpr were not observed in the two animals in which reversion of vpr was not detected. Antibody responses to gag and to whole virus antigens were of similar strength in all seven animals. Both control animals and two of the test animals in which vpr reverted maintained high virus loads and developed progressive disease. Low virus burden and no disease have been observed in the two animals in which vpr did not revert and in the one animal in which vpr reversion was first detected only at 8 weeks. The reversion of vpr in three of the five test animals indicates that there is significant selective pressure for functional forms of vpr in vivo. Furthermore, the results suggest that both vpr and nef are important for maximal SIV replication and persistence in vivo and for disease progression.

Amino Acid Sequence↗

PubMed1993

Restricted replication of simian immunodeficiency virus strain 239 in macrophages is determined by env but is not due to restricted entry.

Virus derived from the infectious, pathogenic, molecular clone of simian immunodeficiency virus (SIV) called SIVmac239 replicates poorly in primary rhesus monkey alveolar macrophage cultures. Variants with three to nine amino acid changes in the envelope replicate 100 to 1,000 times more efficiently in these macrophage cultures than parental SIVmac239. Early events, including virus entry into cells, were analyzed by measuring the amounts of newly synthesized viral DNA 14 to 16 h after infection of macrophages by using a quantitative polymerase chain reaction method. SIVmac239 ws found to enter macrophages with an efficiency similar to that of the macrophage-tropic derivatives. The assay indeed measured newly synthesized viral DNA since detection was inhibited by the reverse transcriptase inhibitors azidothymidine and foscarnet and by heat inactivation of the virus stock prior to infection. Furthermore, entry of SIVmac239 and macrophage-tropic variant into macrophages was inhibited by monoclonal antibody against CD4. Analysis of the time course of viral DNA accumulation showed that although initial entry of SIVmac239 into cells occurred normally, subsequent logarithmic increases in the amounts of viral DNA associated with spread of virus through the macrophage cultures was blocked. Increasing the amount of SIVmac239 incubated with macrophages increased the amount of virus entering the cell, but this could not overcome the block to replication. Thus, restricted replication of SIVmac239 in macrophages is determined by the envelope, but surprisingly it is not due to restricted virus entry.

Animals↗

PubMed1994

A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge.

Three infectious, attenuated molecular clones of simian immunodeficiency virus (SIVmac) were tested for viral and host determinants of protective immunity. The viruses differed in degree of virulence from highly attenuated to moderately attenuated to partially attenuated. Levels of immune stimulation and antiviral immunity were measured in rhesus macaques inoculated 2 years previously with these viruses. Monkeys infected with the highly attenuated or moderately attenuated viruses had minimal lymphoid hyperplasia, normal CD4/CD8 ratios, low levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against p55gag (Gag) or gp160env (Env). Monkeys infected with the partially attenuated virus had moderate to marked lymphoid hyperplasia, normal CD4/CD8 ratios, high levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against both Gag and Env. After pathogenic virus challenge, monkeys immunized with the partially attenuated virus had 100- to 1,000-fold-lower viral load in peripheral blood mononuclear cells and lymph node mononuclear cells than naive control animals. One of four monkeys immunized with the highly attenuated virus and two of four monkeys immunized with the moderately attenuated virus developed similarly low viral loads after challenge. These three attenuated strains of SIV induced a spectrum of antiviral immunity that was inversely associated with their degree of attenuation. Only the least attenuated virus induced resistance to challenge infection in all immunized monkeys.

Animals↗

PubMed1997

A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.

Sequences at the left terminus of U3 in the left long terminal repeat (LTR) and at the right terminus of U5 in the right LTR are important for integration of retroviral DNA. In the infectious pathogenic molecular clone of simian immunodeficiency virus strain mac239 (SIVmac239), 10 of the 12 terminal base pairs form an imperfect inverted repeat structure (5' TGGAAGGGATTT 3' [nucleotides 1 to 12] and 3' ACGATCCCTAAA 5' [nucleotides 10279 to 10268]). Nineteen different mutant forms of SIVmac239 proviral DNA with changes at one or more of the positions in each of the 12-terminal-base-pair regions were constructed. Viral replication was severely or completely compromised with nine of these mutants. Revertants appeared 40 to 50 days after transfection in two independent experiments with mutant 7, which contained changes of AGG to TAC at positions 5 to 7 in U3 and TCC to GAA at positions 10275 to 10273 in U5. Virus produced at these times from mutant 7 transfection replicated upon reinfection with only a slight delay when compared to the wild type. Sequence analysis of the LTR and integrase regions from infected cultures revealed two predominant changes: G to A at position 10275 in U5 and Glu to Lys at position 136 in integrase. Derivatives of clone 7 in which these changes were introduced individually and together were constructed by site-specific mutagenesis. Each change individually restored replication capacity only partially. However, the combination of both mutations restored replicative capacity to that of the original revertants. These results indicate that changes in integrase can compensate for mutations in the terminal nucleotides of the SIV LTR. The results further indicate that resistance to integrase inhibitors may include both integrase and LTR mutations.

Amino Acid Substitution↗

PubMed1997

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.

Anti-HIV Agents↗

PubMed1998

Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors.

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5' splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34(+) CD38(-) human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.

Antigens, CD34↗