The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

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The scanning model for translation: an update.

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.

https://www.pnas.org/content/pnas/83/21/8122.full.pdf

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase

Human immunodeficiency virus type 1 (HIV-1) Env-, Gag-, Pol-, Nef-, and Tat-specific cytotoxic T-lymphocyte (CTL) activities were quantitated temporally in five patients with symptomatic primary HIV-1 infection. A dominant CD8(+)-mediated, major histocompatibility complex class I-restricted CTL response to the HIV-1 envelope glycoprotein, gp160, was noted in four of the five patients studied. The level of HIV-1-specific CTL activity in the five patients paralleled the efficiency of control of primary viremia. Patients who mounted strong gp160-specific CTL responses showed rapid reduction of acute plasma viremia and antigenemia, while in contrast, primary viremia and antigenemia were poorly controlled in patients in whom virus-specific CTL activity was low or undetectable. These results suggest that HIV-1-specific CTL activity is a major component of the host immune response associated with the control of virus replication following primary HIV-1 infection and have important implications for the design of antiviral vaccines.

https://jvi.asm.org/content/jvi/68/9/6103.full.pdf

Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.

It has been proposed that changes in cell surface concentrations of coreceptors may control infections by human immunodeficiency virus type 1 (HIV-1), but the mechanisms of coreceptor function and the concentration dependencies of their activities are unknown. To study these issues and to generate stable clones of adherent cells able to efficiently titer diverse isolates of HIV-1, we generated two panels of HeLa-CD4/CCR5 cells in which individual clones express either large or small quantities of CD4 and distinct amounts of CCR5. The panels were made by transducing parental HeLa-CD4 cells with the retroviral vector SFF-CCR5. Derivative clones expressed a wide range of CCR5 quantities which were between 7.0 x 10(2) and 1.3 x 10(5) molecules/cell as measured by binding antibodies specific for CCR5 and the chemokine [125I]MIP1beta. CCR5 was mobile in the membranes, as indicated by antibody-induced patching. In cells with a large amount of CD4, an unexpectedly low trace of CCR5 (between 7 x 10(2) and 2.0 x 10(3) molecules/cell) was sufficient for maximal susceptibility to all tested HIV-1, including primary patient macrophagetropic and T-cell-tropic isolates. Indeed, the titers as indicated by immunoperoxidase staining of infected foci were as high as the tissue culture infectious doses measured in human peripheral blood mononuclear cells. In contrast, cells with a small amount of CD4 required a much larger quantity of CCR5 for maximal infection by macrophagetropic HIV-1 (ca. 1.0 x 10(4) to 2.0 x 10(4) molecules/cell). Cells that expressed low and high amounts of CD4 were infected with equal efficiencies when CCR5 concentrations were above threshold levels for maximal infection. Our results suggest that the concentrations of CD4 and CCR5 required for efficient infections by macrophagetropic HIV-1 are interdependent and that the requirements for each are increased when the other component is present in a limiting amount. We conclude that CD4 and CCR5 directly or indirectly interact in a concentration-dependent manner within a pathway that is essential for infection by macrophagetropic HIV-1. In addition, our results suggest that multivalent virus-receptor bonds and diffusion in the membrane contribute to HIV-1 infections.

Effects of CCR5 and CD4 Cell Surface Concentrations on Infections by Macrophagetropic Isolates of Human Immunodeficiency Virus Type 1

The complete DNA sequence of vaccinia virus

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

A detailed kinetic and quantitative analysis of the early and late biosynthetic events undergone by the human immunodeficiency virus type 1 envelope protein expressed by a recombinant vaccinia virus was performed. Early folding events that occurred in the endoplasmic reticulum included disulfide bond formation (t1/2 approximately 10 min), folding of envelope protein into a form competent to bind CD4 (t1/2 approximately 15 min), and specific and transient association and dissociation with GRP78-BiP (t1/2 approximately 25 min). After initial folding, envelope protein monomers formed noncovalently associated dimers with high efficiency (t1/2 approximately 30 min). Studies with brefeldin A, a compound that inhibits endoplasmic reticulum-to-Golgi transport, suggested that assembly occurred in the endoplasmic reticulum while cleavage of gp160 into gp120/gp41 subunits occurred in a post-endoplasmic reticulum compartment. Transport to the Golgi was monitored by modification of N-linked sugars to forms partially resistant to endoglycosidase H. The kinetics of endoglycosidase H resistance were nearly identical to the kinetics of gp160 cleavage (t1/2 approximately 80 min). Cleavage efficiency was strongly cell type dependent, ranging from 13 to 70%. By contrast, approximately 50% of the gp120 generated by the cleavage event was shed (t1/2 approximately 120 min) regardless of the cell type used. The results are discussed in terms of the overall biosynthetic pathway of the envelope protein and provide a framework with which to assess the effects of mutations on structure and function.

Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.

The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes.

Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment.

The effects of C-terminal and internal deletions on the synthesis, transport, biological properties, and antigenicity of the human immunodeficiency virus type 1 envelope protein were determined. A family of recombinant vaccinia viruses that express N-terminal overlapping env proteins of 204, 287, 393, 502 (full-length gp120), 635, 747, and 851 (full-length gp160) amino acids was constructed. All of the proteins were detected in intra- and extracellular forms which differed in the extent of glycosylation. The 747- and 851-amino-acid proteins were cleaved, were expressed on the surface of infected cells, and bound CD4. The 635-amino-acid env protein was cleaved inefficiently, and both the precursor and product were secreted, indicating absence of the transmembrane sequence. The 635- as well as the 502-amino-acid protein, which was also largely secreted, could still bind CD4. Unexpectedly, the 393-amino-acid protein was anchored in the plasma membrane, but neither it nor smaller proteins bound to soluble CD4. When amino acids at the gp120-gp41 junction were deleted, proteolytic cleavage of gp160 did not occur. Nevertheless, gp160 was inserted into the plasma membrane and bound soluble CD4. The predominant conserved B-cell epitopes were mapped to gp41 and the C terminus of gp120, whereas cytotoxic T-cell epitopes were distributed throughout the length of the glycoproteins.

Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.

P L Earl

Papers

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.

Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment.

Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses

Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.