bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages
Authors: Vasala A , Dupont L , Baumann M , Ritzenthaler P , Alatossava T
Published: January 2020

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module.
Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation
Authors: Andeweg A C , Leeflang P , Osterhaus A D , Bosch M L
Published: January 2020

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.
An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation
Authors: Pollack J R , Ganem D
Published: January 2020

Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA. By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA. Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop. The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo. These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation.
Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells
Authors: Bagai S , Puri A , Blumenthal R , Sarkar D P
Published: January 2020

Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors.
Dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene
Authors: Blasco R , Sisler J R , Moss B
Published: January 2020

Vaccinia virus strains vary considerably in the amounts of extracellular enveloped virus (EEV) that they release from infected cells. The IHD-J strain produces up to 40 times more EEV than does the related WR strain and consequently generates elongated comet-shaped virus plaques instead of sharply defined round ones in susceptible monolayer cells under liquid medium. The difference in EEV formation is due to the retention of enveloped WR virions on the cell surface (R. Blasco and B. Moss, J. Virol. 66:4170-4179, 1992). By using WR and IHD-J DNA fragments for marker transfer and analyzing the progeny virus by the comet formation assay, we determined that gene A34R and at least one other gene regulate the release of cell-associated virions. Replacement of the A34R gene of WR with the corresponding gene from IHD-J increased the amount of EEV produced by 10-fold and conferred the ability to form distinctive comet-shaped plaques. Gene A34R encodes an EEV-specific glycoprotein with homology to C-type animal lectins (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). The nucleotide sequences of the A34R genes of WR and IHD-J strains differed in six positions, of which four were silent. One of the codon mutations (Lys-151-->Glu), which is located in the putative carbohydrate recognition domain, was sufficient to transfer a comet-forming phenotype to WR virus. These data indicate that the A34R-encoded glycoprotein is involved, through its lectin homology domain, in the retention of progeny virus on the surface of parental cells and raise the possibility that the protein also has a role in virus attachment to uninfected cells.
Inhibition of growth, transformation, and expression of human papillomavirus type 16 E7 in human keratinocytes by alpha interferons
Authors: Khan M A , Tolleson W H , Gangemi J D , Pirisi L
Published: January 2020

We used a model system of normal human keratinocytes (HKc) and HKc immortalized with human papillomavirus type 16 DNA (HKc/HPV16) to investigate the effects of alpha interferons (IFN-alpha) on the growth of HPV16-immortalized human epithelial cells, on HPV16-mediated immortalization of normal HKc, and on HPV16 gene expression. Normal HKc and HKc/HPV16 were treated with several recombinant human IFN-alpha subtypes (IFN-alpha B, IFN-alpha D, and IFN-alpha B/D). These IFN-alpha subtypes inhibited proliferation of both normal HKc and HKc/HPV16 in a dose-dependent fashion; however, although 1,000 to 10,000 U of IFN-alpha per ml were required to inhibit growth of normal HKc, HKc/HPV16 were substantially growth inhibited by 100 U/ml. In addition, 100 U of IFN-alpha B/D per ml inhibited transformation of normal HKc by HPV16 DNA. Northern (RNA) blot analysis showed no effect of IFN-alpha on the mRNA levels of the HPV16 E6 and E7 open reading frames. However, immunofluorescence studies of the HPV16 E6 and E7 proteins with anti-E6 and anti-E7 monoclonal antibodies showed significant inhibition of E7 protein expression in cells treated with IFN-alpha, whereas E6 protein expression was not altered. The inhibition of E7 protein expression in cells treated with IFN-alpha was further confirmed by Western immunoblot analysis. These results suggest that IFN-alpha may inhibit HPV16-mediated transformation of HKc and proliferation of HKc/HPV16 through an inhibition of HPV16 E7 protein expression.
Cross-reactive lysis of human targets infected with prototypic and clinical human immunodeficiency virus type 1 (HIV-1) strains by murine anti-HIV-1 IIIB env-specific cytotoxic T lymphocytes
Authors: Chada S , DeJesus C E , Townsend K , Lee W T , Laube L , Jolly D J , Chang S M , Warner J F
Published: January 2020

To evaluate the ability of murine anti-human immunodeficiency virus type 1 (HIV-1) IIIB env cytotoxic T lymphocytes (CTL) to recognize and lyse HIV-1-infected cells, we have constructed a human cell line (Hu/Dd) expressing both the CD4 receptor and the murine H-2Dd major histocompatibility complex (MHC) class I protein. This cell line can be productively infected with HIV-1 and can also function as a target for murine CD8+, class I MHC-restricted CTL directed against the envelope glycoprotein of HIV-1 IIIB. The ability of BALB/c anti-HIV-1 IIIB env CTL to specifically recognize and lyse Hu/Dd target cells infected with divergent HIV-1 strains was tested by using both prototypic and clinical HIV-1 strains. CTL generated by immunization of mice with syngeneic cells expressing either the native or V3 loop-deleted (delta V3) envelope glycoprotein from HIV-1 IIIB were able to recognize and specifically lyse Hu/Dd target cells infected with the HIV-1 prototypic isolates IIIB, MN, WMJ II, SF2, and CC as well as several HIV-1 clinical isolates. These results demonstrate that CTL determinants for HIV-1 env exist outside the hypervariable V3 region, anti-HIV-1 IIIB env CTL appear to recognize common determinants on diverse HIV-1 strains, and classification of HIV-1 strains based on neutralizing antibody reactivities does not appear to correspond to CTL recognition and lysis. The results suggest that the cell-mediated components of the immune system may have a broader recognition of divergent HIV-1 strains than do the humoral components.
Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants
Authors: Tengelsen L A , Pederson N E , Shaver P R , Wathen M W , Homa F L
Published: January 2020

The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein. Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C. Addison, F. J. Rixon, and V. G. Preston, J. Gen. Virol. 71:2377-2384, 1990; B. A. Pancake, D. P. Aschman, and P. A. Schaffer, J. Virol. 47:568-585, 1983). In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene. The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene. Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene. Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA. Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells. A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein. This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa. The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection. No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.
The adenovirus L3 23-kilodalton proteinase cleaves the amino-terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells
Authors: Chen P H , Ornelles D A , Shenk T
Published: January 2020

Immunofluorescence studies revealed that adenovirus induces a reorganization of the cytokeratin system in lytically infected HeLa cells. At 24 h postinfection, the cytokeratin network began to disassemble into prominent spheroid globules. By 36 h postinfection, host cell lysis occurred, accompanied by the formation of perinuclear cytokeratin clumps and additional spheroid globules. Immunoblots detected 41- and 44-kDa fragments of cytokeratin 18 and reduced levels of cytokeratin 7 at 24 and 36 h postinfection. Cytokeratin proteins isolated from HeLa cells at 36 h postinfection were deficient in filament polymerization. The 41-kDa proteolytic cytokeratin 18-specific fragment was purified, and its amino-terminal sequence was determined to be GGIQNEKETM. These residues correspond to amino acids 74 through 83 of cytokeratin 18, identifying a cleavage site at the junction of the globular head domain and the alpha-helical rod domain. Moreover, this truncation event occurs at a consensus cleavage site for the adenovirus L3 23-kDa proteinase. The temperature-sensitive mutant H2-ts1, which contains a mutation in the proteinase, neither induced cleavage of cytokeratin 18 nor precipitated the formation of spheroid globules during lytic infection at the nonpermissive temperature. The active proteinase is therefore required for cleavage of cytokeratin 18 and morphological rearrangement of the cytokeratins. We suggest that disruptions in the cytokeratin system weaken the mechanical integrity of the cell, thus promoting host cell lysis and release of progeny virions.
Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells
Authors: Barker K A , Hampe A , Stoeckle M Y , Hanafusa H
Published: January 2020

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.
Inhibition of retrovirus-induced disease in mice by camptothecin
Authors: Priel E , Aflalo E , Chechelnitsky G , Benharroch D , Aboud M , Segal S
Published: January 2020

We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase I-specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases.
Effect of linker insertion mutations in the human immunodeficiency virus type 1 gag gene on activation of viral protease expressed in bacteria
Authors: Luban J , Lee C , Goff S P
Published: January 2020

We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.
Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans
Authors: Ansardi D C , Porter D C , Morrow C D
Published: January 2020

Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of poliovirus capsid proteins derived from a nonmyristylated P1 precursor expressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins generated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids.(ABSTRACT TRUNCATED AT 400 WORDS)
The full-length Tat protein is required for TAR-independent, posttranscriptional trans activation of human immunodeficiency virus type 1 env gene expression
Authors: Kim Y S , Panganiban A T
Published: January 2020

Tat is a protein that dramatically increases the expression of all genes expressed from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat through interaction with a cis-acting target sequence referred to as TAR (for trans-acting responsive region). The tat gene is divided into two coding exons which, when translated, result in the synthesis of an 86-amino-acid protein. However, the 72-amino-acid segment encoded by the first coding exon of tat is sufficient to encode a fully active Tat protein in known assays. We examined expression of the env gene from an LTR that lacks TAR (designated dTAR-env). Surprisingly, only the full-length Tat peptide trans activated expression of the env gene from dTAR-env. Comparison of RNA and protein expression of the env gene in the presence of Tat indicated that the mechanism of trans activation is posttranscriptional rather than transcriptional. To test whether the TAR-independent Tat function is specific to the HIV-1 env gene, we analyzed expression of heterologous genes from the long terminal repeat lacking TAR. These heterologous genes were not trans activated by Tat in the absence of a TAR element, which suggests that the second-exon peptide of Tat has a sequence-specific role in TAR-independent trans activation of the HIV-1 env gene. Analysis of a mutant in the 5' end of the env gene was used to identify a cis-acting sequence required for Tat responsiveness.
A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice
Authors: DiFronzo N L , Holland C A
Published: January 2020

We analyzed viral recombination events that occur during the preleukemic period in AKR mice. We tagged a molecular chimera between the nonleukemogenic virus Akv and the leukemogenic mink cell focus-inducing (MCF) virus MCF 247 with an amber suppressor tRNA gene, supF. We injected the supF-tagged chimeric virus that contains all of the genes of MCF 247 except the envelope gene, which in turn is derived from Akv, into newborn AKR mice to evaluate its pathogenic potential. Approximately the same percentage of animals developed leukemia with similar latent periods when injected with either the tagged or nontagged virus. DNA from tumors induced in AKR mice by the tagged chimeric virus was analyzed by Southern blotting with the supF gene as a probe. One set of tumors contained the injected supF-tagged virus. Two kinds of supF-tagged proviruses were found in a second set of tumors. One group of supF-tagged viruses had a restriction map consistent with that of the injected virus, while the other group of proviruses had restriction maps that suggested that the proviruses had acquired an MCF virus-like envelope gene by recombination with endogenous viral sequences. These results demonstrate that injected viruses recombine in vivo with endogenous viral sequences. Furthermore, the progression to leukemia was accelerated in mice that develop tumors containing proviruses with an MCF virus env gene, emphasizing the importance of the role of the MCF virus env gene product in transformation.
Functional interaction of nuclear factors EF-C, HNF-4, and RXR alpha with hepatitis B virus enhancer I
Authors: Garcia A D , Ostapchuk P , Hearing P
Published: January 2020

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to the GB element in vitro. HNF-4 transactivates a promoter linked to a multimerized GB/EF-C domain via the GB element in vivo in a manner that is dependent on the integrity of the adjacent EF-C binding site. RXR alpha also transactivates promoter expression via the GB element in vivo in response to retinoic acid but in a largely EF-C-independent manner. Finally, we show that COUP-TF antagonizes the activity of the GB element in human liver cells.
Independent variation and positive selection in env V1 and V2 domains within maternal-infant strains of human immunodeficiency virus type 1 in vivo
Authors: Lamers S L , Sleasman J W , She J X , Barrie K A , Pomeroy S M , Barrett D J , Goodenow M M
Published: January 2020

Multiple targets for immune recognition and cellular tropism are localized to the V1 and V2 hypervariable regions in the amino portion of human immunodeficiency virus type 1 (HIV-1) gp120env. We have assessed genetic diversity in env V1 and V2 hypervariable domains in vivo within epidemiologically related strains of HIV-1. Our strategy was to analyze longitudinal samples from two seropositive mothers and multiple children infected by perinatal transmission. Although the V1 and V2 domains are closely linked in the HIV-1 genome, nucleotide sequences in V1 and in V2 evolved independently in maternal-infant viruses in vivo. A high proportion of the nucleotide substitutions would introduce amino acid diversity in V1 and in V2. A significant excess of nonsynonymous over synonymous substitutions was identified in HIV-1 env V1 and V2 peptides in the mothers and in two older children but was not generally apparent in HIV-1 sequences in infants. An excess of nonsynonymous over synonymous substitutions indicated that there is positive selection for independent genetic variation in the V1 and V2 domains in vivo. It is likely that there are host responses to complex determinants in the V1 or V2 hypervariable domain of HIV-1 gp120.
NS3 is a serine protease required for processing of hepatitis C virus polyprotein
Authors: Tomei L , Failla C , Santolini E , De Francesco R , La Monica N
Published: January 2020

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.
Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain
Authors: Smith J S , Roth M J
Published: January 2020

We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.
Vaccination to prevent persistent viral infection
Authors: Oldstone M B , Tishon A , Eddleston M , de la Torre J C , McKee T , Whitton J L
Published: January 2020

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.
Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages
Authors: Balotta C , Lusso P , Crowley R , Gallo R C , Franchini G
Published: January 2020

The replication of human immunodeficiency viruses (HIV) in human macrophages is influenced by genetic determinants which have been mapped predominantly to the viral envelope. However, in HIV-2, the vpr gene has also been suggested as an important modulator of viral expression in human macrophages. We synthesized five antisense phosphorothioate oligodeoxynucleotides complementary to the vpr mRNA of HIV-1Ba-L, a highly macrophage-tropic viral strain, and measured their effect on HIV-1Ba-L replication in primary human macrophages. All of the oligodeoxynucleotides displayed some level of non-sequence-specific inhibition of viral replication; however, only the antisense one had an additional effect on viral production in primary macrophages. Of the five antisense oligodeoxynucleotides tested, only one did not show any additional effect on viral production, whereas all the others inhibited viral replication to a similar degree (70 to 100%). Variation in the degree of inhibition was observed by using five different donors of human primary macrophages. The phosphorothioate oligonucleotides, targeted to the initiating methionine of the Vpr protein, had an inhibitory effect at both 20 and 10 microM only when the size was increased from 24 to 27 bases. Thus, HIV-1 replication in human macrophages is modulated by the expression of the vpr gene, and it is conceivable that vpr antisense oligodeoxynucleotides could be used in combination with antisense oligodeoxynucleotides against other HIV-1 regulatory genes to better control viral expression in human macrophages.
African swine fever virus encodes a serine protein kinase which is packaged into virions
Authors: Baylis S A , Banham A H , Vydelingum S , Dixon L K , Smith G L
Published: January 2020

Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.
Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein
Authors: Mangold C M , Streeck R E
Published: January 2020

The small envelope protein of hepatitis B virus is the major component of the viral coat and is also secreted from cells as a 20-nm subviral particle, even in the absence of other viral proteins. Such empty envelope particles are composed of approximately 100 copies of this polypeptide and host-derived lipids and are stabilized by extensive intermolecular disulfide cross-linking. To study the contribution of disulfide bonds to assembly and secretion of the viral envelope, single and multiple mutants involving all 14 cysteines in HepG2 and COS-7 cells were analyzed. Of the six cysteines located outside the region carrying the surface antigen, Cys-48, Cys-65, and Cys-69 were each found to be essential for secretion of 20-nm particles, whereas Cys-76, Cys-90, and Cys-221 were dispensable. By introduction of an additional cysteine substituting serine 58, the yield of secreted particles was increased. Of four mutants involving the eight cysteines located in the antigenic region, only the double mutant lacking Cys-121 and Cys-124 was secreted with wild-type efficiency. Secretion-competent envelope proteins were intracellularly retained by secretion-deficient cysteine mutants. According to alkylation studies, both intracellular and secreted envelope proteins contained free sulfhydryl groups. Disulfide-linked oligomers were studied by gel electrophoresis under nonreducing conditions.
Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies
Authors: Pfeiffer B , Berneman Z N , Neipel F , Chang C K , Tirwatnapong S , Chandran B
Published: January 2020

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.
Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo
Authors: Shugars D C , Smith M S , Glueck D H , Nantermet P V , Seillier-Moiseiwitsch F , Swanstrom R
Published: January 2020

The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.
Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4
Authors: Patton J T , Hua J , Mansell E A
Published: January 2020

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.
A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum
Authors: Fäcke M , Janetzko A , Shoeman R L , Kräusslich H G
Published: January 2020

Morphogenesis of retroviruses involves assembly of the structural Gag and Gag-Pol polyproteins with subsequent budding of the virus particle from the plasma membrane and proteolytic cleavage by the viral proteinase. The matrix (MA) domain, representing the N-terminal segment of Gag, plays a critical role in this process. We constructed an in-frame deletion in the MA coding region (lacking codons 16 to 99) of the human immunodeficiency virus (HIV) type 1 gag gene. Following transient transfection of the complete proviral DNA carrying the deletion, the mutant polyprotein was synthesized and proteolytically processed like the wild-type polyprotein. However, release of virus particles was reduced approximately 10-fold. The extracellular particles that were released did not contain viral glycoproteins and were noninfectious. Electron micrographs revealed budding of virus particles into the endoplasmic reticulum (ER) of transfected cells and large numbers of particles within the ER. These particles were all immature and morphologically indistinguishable from intracisternal A-type particles, a class of murine endogenous retrovirus elements. Budding structures at the plasma membrane were rarely seen and only a few extracellular particles were observed, but in contrast to those in the ER, these particles had the morphology of mature particles, similar to that of wild-type HIV, except for the lack of surface projections.
Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins
Authors: Furnari B A , Poma E , Kowalik T F , Huong S M , Huang E S
Published: January 2020

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augmented by immediate-early gene product 1 (IE1). IE2 has also been shown to autoregulate the major immediate-early promoter by directly binding to a cis repression signal located between the TATA box and the cap site. However, IE2 has not been shown to act directly through a specific DNA sequence in transactivating various promoters. To understand whether IE2 can be indirectly involved in DNA sequence-specific transactivation through interactions with other transcriptional factors, we performed a study of the interactions of IE2 with cellular proteins. In order to study these interactions, IE cDNAs were subcloned into a bacterial expression vector, pGEX2T, by polymerase chain reaction amplification to produce fusion proteins which were full-length as well as proteins which contained various functional domains. We were able to demonstrate IE2's ability to interact directly or indirectly with several cellular proteins ranging from > 200 to 14 kDa through glutathione S-transferase-fusion protein precipitation and far-Western analysis. These interactions have been mapped to domains within IE2 which are known to be necessary for either transactivation or both transactivation and autoregulation. All of the IE2-associated proteins are nuclear proteins, and a subset are phosphorylated. In vitro-synthesized 35S-IE2 protein and bacterially expressed IE2 fusion proteins were used to study IE2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 interactions were mapped to a domain containing a putative helix-turn-helix motif located near the C terminus of IE2, between amino acids 456 and 539. However, IE2 was unable to directly interact with either IE1, an alternatively spliced variant of IE2 (55 kDa), or IE2 deletion mutants that did not contain the multimerization domain.
TAR loop-dependent human immunodeficiency virus trans activation requires factors encoded on human chromosome 12
Authors: Hart C E , Galphin J C , Westhafer M A , Schochetman G
Published: January 2020

The trans-activator response region (TAR) RNA in the human immunodeficiency virus type 1 (HIV-1) and HIV-2 long terminal repeat forms stem-loop secondary structures in which the loop sequence is essential for trans activation. We investigated how the HIV trans-activation mechanism encoded on human chromosome 12 relates to the TAR RNA loop-dependent pathway. DNA transfection experiments showed that trans activation in human-hamster hybrid cells with the single human chromosome 12 and human T-cell lines was highly dependent on the native sequences of the HIV-1 TAR loop and the HIV-2 5' TAR loop. In nonhuman cell lines or hybrid cells without chromosome 12 that supported trans activation, the cellular mechanism was independent of the HIV-1 TAR loop and the response to mutations in the HIV-2 TAR loops differed from that found in human T-cell lines and human-hamster hybrid cells with chromosome 12. Our results suggest that the human chromosome 12 mechanism interacts directly with the TAR RNA loop or indirectly by regulating TAR RNA-binding proteins.