Showing papers in "Journal of Virology in 1993"

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Abstract: Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1.

Abstract: Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1 In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5 Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS

Expression and identification of hepatitis C virus polyprotein cleavage products.

Abstract: Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, E1 (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82- to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of E1 is associated with E2 and E2-NS2 via disulfide linkages.

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

Abstract: The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.

Abstract: A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

Abstract: The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

Abstract: During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Abstract: Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.

Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.

Abstract: Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

Abstract: The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection. Images

Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.

Abstract: We have studied processing of the nonstructural (NS) polyprotein of the hepatitis C virus. A series of cDNAs corresponding to predicted NS2/3/4 or NS3/4 regions were constructed, and processing of the polyproteins was studied in an in vitro transcription-translation system. We report that a catalytically active serine-type proteinase is encoded by the NS3 region. Substitution of the serine residue of the putative catalytic triad (H, D, and S) by alanine blocked cleavage at the NS3/4 junction, while processing between NS2 and NS3 was not affected. Thus, cleavage at the NS2/3 junction is mediated either by cellular enzymes or by an NS-2 inherent proteinase activity. Deletion analysis of an NS3/4 cDNA construct mapped the amino terminus of the enzymatically active proteinase between amino acids 1049 and 1065 of the polyprotein. As internal deletions of variable segments of the presumed helicase domain prevented processing at the NS314 junction, a continuous NS3 region appears to be required for processing at this site. To analyze hepatitis C virus polyprotein cleavage in vivo, recombinant vaccinia viruses expressing NS2/3/4 or NS3/4/5 proteins were generated. In agreement with the in vitro data, cleavage between NS2 and NS3 was independent of a catalytically active NS3 proteinase, whereas substitution of the active-site serine residue by the amino acid alanine completely blocked processing at the NS3/4 and NS4/5 junctions. These results demonstrate that NS3 encodes the viral proteinase essential for generating the amino termini of NS4 and NS5.

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

Abstract: When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.

NS3 is a serine protease required for processing of hepatitis C virus polyprotein.

Abstract: Hepatitis C virus (HCV) possesses a positive-sense RNA genome which encodes a large polyprotein of 3,010 amino acids. Previous data and sequence analysis have indicated that this polyprotein is processed by cellular proteases and possibly by a virally encoded serine protease localized in the N-terminal domain of nonstructural protein NS3. To characterize the molecular aspects of HCV protein biogenesis and to clearly identify the protein products derived from the HCV genome, we have examined HCV polyprotein expression by using the vaccinia virus T7 transient expression system in transfected cells and by cell-free translation studies. HCV proteins were identified by immunoprecipitation with region-specific antisera. Here we show that the amino-terminal region of the HCV polyprotein is processed in vitro by cellular proteases releasing three structural proteins: p21 (core), gp37 (E1), and gp61 (E2). Processing of the nonstructural region of HCV was evident in transfected cells. Two proteins of 24 and 68 kDa were immunoprecipitated with anti-NS2 and NS3 antisera, respectively. Antiserum against NS4 recognized three proteins of 6, 26, and 31 kDa, while antisera specific for NS5 immunoprecipitated two polypeptides of 56 and 65 kDa, indicating that each of these two genes encodes at least two different proteins. When the NS3 protease domain was inactivated by replacing the proposed catalytic Ser-1165 with Ala, processing at several sites was abolished. When Ser-1164 was mutated to Ala, no effect on the processing was observed. Cleavage activities at three of the four sites affected by NS3 were shown to occur in trans, while processing at the carboxy terminus of NS3 could not be mediated in trans. These results provide a detailed description of the protein products obtained from the processing of the HCV polyprotein. Furthermore, the data obtained implicate NS3 as a serine protease and demonstrate that a catalytically active NS3 is necessary for cleavage of the nonstructural region of HCV.

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Abstract: Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

Abstract: The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Abstract: 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 rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.

Abstract: The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from cloned genes when the DI RNA was synthesized from plasmid. Limited nucleotide additions or deletions (+7 to -7 nucleotides) in the DI RNA sequence were then made at five different sites, and the different RNA derivatives were tested for their abilities to replicate. Efficient replication was observed only when the total nucleotide number was conserved, regardless of the modifications, or when the addition of a total of 6 nucleotides was made. The replicated RNAs were shown to be properly enveloped into virus particles. It is concluded that, to form a proper template for efficient replication, the Sendai virus RNA must contain a total number of nucleotides which is a multiple of 6. This was interpreted as the need for the nucleocapsid protein to contact exactly 6 nucleotides.

Packaging capacity and stability of human adenovirus type 5 vectors.

Abstract: Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.

Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus.

Abstract: Gene products of hepatitis C virus (HCV), a possible major causative agent of posttransfusion non-A, non-B hepatitis, are considered to be produced from a precursor polyprotein via proteolytic processing mediated by either host cell or viral proteinases. The presence of HCV serine proteinase has been proposed from analyses of amino acid sequence homology. To examine the processing mechanism of the HCV precursor polyprotein, the amino-terminal region of the putative nonstructural protein region of the HCV genome, containing the serine proteinase motif, was expressed and analyzed by using an in vitro transcription/translation system and a transient expression system in cultured cells. Two distinct proteinase activities which function in the production of a 70-kDa protein (p70) from the precursor polyprotein were detected. One of these proteinase activities, which cleaved the carboxyl (C)-terminal side of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested that the predicted HCV serine proteinase was functional. The other activity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From the results of pulse-chase analysis, using proteinase inhibitors coupled with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.

La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate.

Abstract: Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjogren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.

Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells.

Abstract: The human immunodeficiency virus type 1 (HIV-1) vif gene encodes a 23-kDa protein of unknown function, also produced by most other known lentiviruses. Vif was found to be essential for the spread of HIV-1 in peripheral blood lymphocytes and in primary macrophages, as well as in some but not all established T-cell lines. Vif was required at the stage of viral particle formation, for cell-to-cell as well as for cell-free transmission of HIV-1. Accordingly, vif-defective viruses could be complemented by the expression of vif in the producer but not in the target cell. vif-defective virions contained wild-type amounts of Gag and Env proteins, reverse transcriptase, integrase, genomic RNA, and partial reverse transcripts. Most importantly, they could enter cells normally, and the vif defect could not be rescued through the use of HIV(MLV [murine leukemia virus]) pseudotypes. Instead, vif-mutant viruses were severely impaired in their ability to complete the synthesis of proviral DNA, once internalized in the target cell. These results suggest that Vif plays a role which is novel for a retroviral protein, in allowing the processing and/or the transport of the internalized HIV core.

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Abstract: Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.

Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.

Abstract: The rate of clinical progression is variable among individuals infected with human immunodeficiency virus type 1 (HIV-1). Changes in viral burden which correlate with disease status have been demonstrated in cross-sectional studies; however, a detailed longitudinal study of the temporal relationship between viral burden, CD4+ T-cell numbers, and clinical status throughout the course of infection has not been reported. Multiple longitudinal blood samples were obtained from four HIV-1-infected individuals with clinically divergent profiles. Levels of HIV-1 were measured in sequential samples of peripheral blood mononuclear cells, using both end-point dilution cultures and quantitative polymerase chain reaction methods. Serial HIV-1 isolates from each case were also evaluated to determine their biological properties in vitro. For the three patients with clinical progression, a dramatic increase in the level of HIV-1 was observed concurrent with or prior to a marked drop in CD4+ T lymphocytes. This increase in viral burden was temporally associated with the emergence of a more cytopathic viral phenotype. In contrast, consistently low levels of HIV-1 were observed in the one patient who was clinically and immunologically stable for more than a decade. Moreover, viral isolates from this patient were less cytopathic in vitro compared with HIV-1 isolates from those patients with disease progression. The temporal association between increased viral burden and CD4+ T-cell decline suggests a direct role for HIV-1 in the cytopathology of CD4+ T cells in vivo. Our results indicate that the pathogenic mechanisms responsible for CD4+ T-cell depletion may be related to both quantitative and qualitative changes in HIV-1.

Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism.

Abstract: The human hepatitis C virus (HCV) contains a long 5' noncoding region (5' NCR). Computer-assisted and biochemical analyses suggest that there is a complex secondary structure in this region that is comparable to the secondary structures that are found in picornaviruses (E.A. Brown, H. Zhang, L.-H. Ping, and S.M. Lemon, Nucleic Acids Res. 20:5041-5045, 1992). Previous in vitro studies suggest that the HCV 5' NCR plays an important role during translation (K. Tsukiyama-Kohara, N. Iizuka, M. Kohara, and A. Nomoto, J. Virol. 66:1476-1483, 1992). Dicistronic and monocistronic expression vectors, in vitro translation, RNA transfections, and deletion mutagenesis studies were utilized to demonstrate unambiguously that the HCV 5' NCR is involved in translational control. Our data strongly support the conclusion that an internal ribosome entry site exists within the 5' noncoding sequences proximal to the initiator AUG. Furthermore, our results suggest that the HCV genome is translated in a cap-independent manner and that the sequences immediately upstream of the initiator AUG are essential for internal ribosome entry site function during translation.

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins.

Abstract: Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.

Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.

Abstract: Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.

Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation.

Abstract: Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or -3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformation-defective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation.

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

Abstract: The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.

Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis.

Abstract: The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication.

Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA.

Abstract: Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination. Images