We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified. * Corresponding author. MATERIALS AND METHODS Cells. The cell lines used in these studies are listed in Table 10 [ig of uncleaved plasmid DNA in each assay. Virus production was monitored in non-T4 cells by cocultivation with CD4+ A3.01 cells (106 cells of each type) 2 days after transfection. Reverse transcriptase (RT) assays were carried 284

Virus-specific antibodies protect individuals against a wide variety of viral infections. To assess whether human immunodeficiency virus type 1 (HIV-1) envelope-specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV-1 isolates, and used this for passive immunization of pig-tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian-human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV-1DH12, a dual-tropic primary virus isolate. Here we show that anti-SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody-mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV-challenged macaques was in the range of 1:5-1:8. The HIV-1-specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non-neutralizing anti-HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

The synthesis and processing of the human immunodeficiency virus 1 (HIV-1) envelope precursor glycoprotein gpl60 was studied in an infected CD4' lymphocytic cell line. Surprisingly, only a small percentage (5-15%) of gpl60 is cleaved to produce the mature gpl20 component. Intracellular sorting results in the transfer of most uncleaved gpl60 to lysosomes, where it is degraded, while gpl20 is transported to the cell surface and subsequently secreted. Cleavage of gpl6O to generate gpl20 occurs intracellularly and can be inhibited by NH4Cl. Taken together, these results indicate that intracellular cleavage of gpl60 determines the intracellular transport and survival of the envelope glycoproteins necessary to produce infectious virus.Human immunodeficiency virus 1 (HIV-1) is a human retrovirus and the primary etiological agent associated with the acquired immunodeficiency syndrome (AIDS). Like other retroviruses, the HIV-1 genome encodes envelope glycoproteins, which project from the membrane surface of mature particles. The external glycoproteins of several viruses are synthesized as precursor molecules, which are ultimately processed to mature proteins that mediate functions required for infection (1). These include the gp70/pl5E envelope gene products of the murine retroviruses, the HA1/HA2 hemagglutinin proteins of the influenza virus, and the F1/F2 fusion proteins of Sendai virus (1, 2). The same is true for HIV-1, in which the gpl60 glycoprotein precursor is cleaved to form the external gpl20 and gp4l membrane-associated envelope components (3, 4). The cleavage of gpl60 is essential for HIV-1 infectivity (5). Functional studies indicate that gpl20 is responsible for the adsorption of virions to the CD4 receptor (6, 7), while gp4l mediates the fusion of viral and cellular membranes (8,9). Expression of HIV-1 envelope proteins on the surface of infected cells can also lead to cell-to-cell fusion, resulting in the formation of syncytia (10).While information continues to accumulate pertaining to the functional characterization of the envelope glycoproteins, the intracellular synthesis and processing of these proteins is not well understood. In this study we have quantitatively analyzed the fate of newly synthesized gpl60 during productive HIV-1 infection of CD4+ cells. Pulsechase analyses indicate that only a small fraction of the precursor gpl60 is processed into the mature gpl20 component. The uncleaved gpl60 is efficiently transported to and degraded within lysosomes, while a greater proportion of the gpl20 remains stable and is eventually secreted from the cell.MATERIALS AND METHODS Cell Cultures. A3.01, a human lymphocytic leukemia cell line (11), was maintained in RPMI 1640 medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (RPMI/FCS). Peripheral blood lymphocytes were grown in RPMI/FCS medium containing 0.25 ,ug of phytohemagglutinin per ml for 3 days prior to infection. The cells were then transferred to and maintained in RPMI/FCS medium containing human interleukin...

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gpl60. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gpl60 to gpl20 and gp4l. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gpl60 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gpl60, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gpl60. The involvement of gpl60 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu. * Corresponding author. MATERIALS AND METHODS Cells and transfections. HeLa cells (ATCC CCL2) were propagated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For transfection, HeLa cells were grown to near confluency in 25-cm2 flasks (3 x 106 cells per flask). Before transfection, the medium was replaced with 5 ml of fresh DMEM containing 10% FBS. Calcium phosphate-precipitated (10) plasmid DNA (4 to 30 pLg) was added to the cells. After 3 h, the medium was removed, and the cells were subjected to a glycerol shock for 2.5 min (9). The cultures were then washed once with phosphate-buffered saline (PBS) and maintained in 5 ml of DMEM containing 10% FBS. Cloned DNAs. All plasmids containing HIV-1 sequences are derivatives of the infectious molecular clone pNL4-3 (1). The plasmid pNL-Al (vift vpr+ tat+ rev+ vpu+ env+ nef) was constructed as described previously by substituting a BssHII-EcoRI fragment from the vif cDNA clone pUCl-11

Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gpl20 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gpl20 and gp4l or the binding of gpl20 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4 + HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gpl20 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gpl20.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gpl60, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gpl60 into the mature virion components, gpl20 and gp4l. We found that coexpression of gpl60 and human CD4 in HeLa cells severely impaired gpl20 production due to the formation of intracellular gpl60-CD4 complexes. This CD4-mediated inhibition of gpl60 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gpl60 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gpl60-CD4 complexes.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.

The nucleotide sequences of the envelope genes of an African and a North American acquired immunodeficiency syndrome (AIDS) viral isolate have been determined. When their deduced amino acid sequences were aligned with the-envelopes of the lymphoadenopathy and AIDS-associated retrovirus isolates, conserved and divergent regions were readily identified. (4,10,11). For example, sequence analysis of the env region of the lymphoadenopathy virus (LAV) and AIDSassociated retrovirus (ARV) isolates revealed the presence of numerous nucleotide substitutions and several reciprocal insertions and-deletions resulting in 9% polynucleotide sequence heterology and a 15% difference in the deduced amino acid sequence (10).The AIDS retrovirus interacts with the immune system in a puzzling way. Virtually everyone infected with the virus synthesizes an antibody directed against a portion of the viral envelope (11-14). However, unlike antibodies reactive with other viral envelopes, the one detected in the sera of individuals exposed to the AIDS retrovirus has little if any protective value (15). Since several therapeutic and preventative strategies of viral intervention currently focus on envelope proteins, a definitive evaluation of the structurally diverse env gene and its polypeptide products is urgently needed. In this report we present the nucleotide and deduced amino acid sequences ofthe env genes of one African and one North American AIDS retrovirus isolate. Their alignment with the analogous segment of the LAV and ARV isolates indicates the location of highly conserved and profoundly divergent domains of the env gene. MATERIALS AND METHODSVirus holates and Molecular Cloning. The African (Z3) and North American (NY5) AIDS retroviruses used in this study were isolated in 1983 and 1984, respectively, and have been previously described (5). Molecular clones of proviral DNAs were obtained from cellular DNA preparations isolated from infected, phytohemagglutinin-stimulated, normal peripheral blood lymphocytes (16). On the basis of previous restriction mapping (5), integrated proviral DNA of NY5 was cloned as two separate EcoRI restriction fragments. Infected cellular DNA was prepared as previously described (17), digested with EcoRI, ligated to EcoRI-digested X Charon 4A (18) arms with T4 DNA ligase, packaged in vitro, and plated on Escherichia coli DP50 supF as previously described (19). The Z3 clone was obtained from a library prepared from a size-selected partial Mbo I digest of infected cellular DNA, inserted into BamHI-cleaved EMBEL-3 (20) phage DNA, packaged in vitro, and plated on E. coli NM539 cells (Promega Biotec, Madison, WI). Approximately 2 x 106 phage plaques from each library were screened (21), using the 2P-labeled pBENN-6 (16) DNA clone of the LAV provirus.Restriction maps of the proviral DNA clones were constructed by using Southern blot hybridization (22) and several contiguous 32P-labeled LAV probes as previously outlined (5). Presumptive full-length proviral DNAs were inserted into pBR322 (NY5 as two s...