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PubMed1995

The highly conserved aspartic acid residue between hypervariable regions 1 and 2 of human immunodeficiency virus type 1 gp120 is important for early stages of virus replication.

Between hypervariable regions V1 and V2 of human immunodeficiency virus type 1 (HIV-1) gp120 lies a cluster of relatively conserved residues. The contribution of nine charged residues in this region to virus infectivity was evaluated by single-amino-acid substitutions in an infectious provirus clone. Three of the HIV-1 mutants studied had slower growth kinetics than the wild-type virus. The delay was most pronounced in a mutant with an alanine substituted for an aspartic acid residue at position 180. This aspartic acid is conserved by all HIV-1 isolates with known nucleotide sequences. Substitutions with three other residues at this position, including a negatively charged glutamic acid, all affected virus infectivity. The defect identified in these mutants suggests that this aspartic acid residue is involved in the early stages of HIV-1 replication.

Amino Acid Sequence↗

PubMed1995

An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.

Base Sequence↗

PubMed1995

Selective infection of human T-lymphotropic virus type 1 (HTLV-1)-infected cells by chimeric human immunodeficiency viruses containing HTLV-1 tax response elements in the long terminal repeat.

Previous studies have suggested that the human immunodeficiency virus long terminal repeat (HIV LTR) enhancer/promoter sequences contribute to the replication ability of HIV in different T-cell lines; mutation of these sequences can alter HIV tropism. We have utilized site-specific mutagenesis to generate variants of HIV that exhibit specific tropism for human T-lymphotropic virus type 1 (HTLV-1) Tax-expressing CD4+ T cells. The wild-type HIV LTR NF-kappa B and Sp1 sites in an infectious molecular clone of HIV type 1 were replaced with sequences derived from the 21-bp Tax response elements (TRE) from the HTLV-1 LTR to generate TRE-containing chimeric HIVs (TRE-HIVs). The TRE-HIVs exhibit selective replication and cell killing in HTLV-infected human CD4+ T cells, but not in HTLV-negative T cells. Transient transfections suggested that Tax-TRE interactions could account for the observed replication specificity. The TRE-containing HIV LTRs were synergistically activated by the HIV Tat and HTLV-1 Tax transactivators. These results demonstrate that it is possible to specifically target HIV replication and cytotoxicity to HTLV-1+, CD4+ human T cells, on the basis of Tax-TRE interactions, and provide a model for the development of specific, cytotoxic, retroviral gene therapy vectors for HTLV-1-infected cells based on alterations of the LTR transcriptional regulatory elements. They also suggest that HIV Tat can cooperate with heterologous transcriptional activators, such as Tax, which act through upstream binding sites without directly binding to DNA.

Base Sequence↗

PubMed1995

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.

Cell Line↗

PubMed1995

Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis.

Coxsackie B virus protein 2B contains near its C terminus a hydrophobic domain with an amino acid composition that is characteristic for transmembrane regions. A molecular genetic approach was followed to define the role of this domain in virus reproduction and to study the structural and hydrophobic requirements of the domain. Nine substitution mutations were introduced in an infectious cDNA clone of coxsackie B3 virus. The effects of the mutations were studied in vivo by transfection of Buffalo green monkey cells with copy RNA transcripts. The results reported here suggest that a critical degree of hydrophobicity of the domain is essential for virus growth. The mutations S77M, C75M, I64S, and V66S, which caused either a small increase or decrease in mean hydrophobicity, yielded viable viruses. The double mutations S77M/C75M and I64S/V6-6S, which caused a more pronounced increase or decrease in hydrophobicity, were nonviable. Negatively charged residues (mutations A71E, I73E, and A71E/I73E) abolished virus growth. The mutations had no effect on the synthesis and processing of the viral polyprotein. Replication and complementation were studied by using a subgenomic coxsackievirus replicon containing the luciferase gene in place of the capsid coding region. Analysis of luciferase accumulation demonstrated that the mutations cause primary defects in viral RNA synthesis that cannot be complemented by wild-type protein 2B provided in trans. The hydrophobic domain is predicted by computer analysis to form a multimeric transmembrane helix. The proposed interaction with the membrane and the implications of the mutations on this interaction are discussed.

Amino Acid Sequence↗

PubMed1995

Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly.

The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.

Animals↗

PubMed1995

Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes.

Base Sequence↗

PubMed1995

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.

Animals↗

PubMed1995

Theiler's virus as a vector for foreign gene delivery.

DA strain and other strains of the TO subgroup of Theiler's murine encephalomyelitis viruses are members of the Cardiovirus genus of picornaviruses and produce a persistent demyelinating disease in mice. A recent study from our laboratory (W.-P. Kong, G. D. Ghadge, and R. P. Roos, Proc. Natl. Acad. Sci. USA 91:1796-1800, 1994) demonstrated that the leader, which is encoded at the N terminus of the Theiler's murine encephalomyelitis virus polyprotein, can be partially replaced by foreign sequences as well as completely deleted, with no loss of infectivity in BHK-21 cells. In this study, we have inserted up to 724 nucleotides into the leader coding region of an infectious DA clone. Recombinant viruses were produced, and the inserts were shown to be stable for at least three passages in BHK-21 cells.

Amino Acid Sequence↗

PubMed1995

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.

Animals↗

PubMed1996

Single amino acid substitution in constant region 1 or 4 of gp120 causes the phenotype of a human immunodeficiency virus type 1 variant with mutations in hypervariable regions 1 and 2 to revert.

The second major cysteine loop of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains 5 to 11 consensus N-linked glycosylation sites, which is disproportionately higher than the number of such sites found in other regions of gp120. Amino acid substitutions introduced at three of six N-linked glycosylation sites in this region of an infectious molecular clone, HXB2, resulted in severe impairment of virus infectivity. Isolation and genetic characterization of a revertant of this mutant revealed an isoleucine-for-valine substitution at position 84 in constant region 1 and an isoleucine-for-methionine substitution at position 434 in constant region 4. Further mutational analysis indicated that either isoleucine substitution was sufficient to confer the revertant phenotype. These findings demonstrate that V1/V2 not only functionally interacts with C4, as previously reported, but also interacts with C1. The observation that compensatory changes do not involve regeneration of N-linked glycosylation sites in the second major cysteine loop suggests that replication of human immunodeficiency virus type 1 in vitro is independent of the presence of a disproportionate number of N-linked glycosylation sites within this loop.

Amino Acid Sequence↗

PubMed1996

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery.

Amino Acid Sequence↗

PubMed1996

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.

Base Sequence↗

PubMed1996

Sequences regulating tropism of human immunodeficiency virus type 1 for brain capillary endothelial cells map to a unique region on the viral genome.

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.

Adult↗

PubMed1996

Coxsackie B3 virus protein 2B contains cationic amphipathic helix that is required for viral RNA replication.

Enterovirus protein 2B has been shown to increase plasma membrane permeability. We have identified a conserved putative amphipathic alpha-helix with a narrow hydrophilic face and an arrangement of cationic residues that is typical for the so-called lytic polypeptides. To examine the functional and structural roles of this putative amphipathic alpha-helix, we have constructed nine coxsackie B3 virus mutants by site-directed mutagenesis of an infectious cDNA clone. Six mutants contained substitutions of the charged residues in the hydrophilic face of the alpha-helix. Three mutants contained insertions of leucine residues between the charged residues, causing a disturbance of the amphipathic character of the alpha-helix. The effect of the mutations on virus viability was assayed by transfection of cells with copy RNA transcripts. The effect on positive-strand RNA replication was examined by introduction of the mutations in a subgenomic luciferase replicon and analysis of luciferase accumulation following the transfection of BGM cells with RNA transcripts. It is shown that both the amphipathy of the domain and the presence of cationic residues in the hydrophilic face of the alpha-helix are required for virus growth. Mutations that disturbed either one of these features caused defects in viral RNA synthesis. In vitro translation reactions and the analysis of viral protein synthesis in vivo demonstrated that the mutations did not affect synthesis and processing of the viral polyprotein. These results suggest that a cationic amphipathic alpha-helix is a major determinant for a function of protein 2B, and possibly its precursor 2BC, in viral RNA synthesis. The potential role of the amphipathic alpha-helix in the permeabilization of cellular membranes is discussed.

Amino Acid Sequence↗

PubMed1996

cis-acting elements in human immunodeficiency virus type 1 RNAs direct viral transcripts to distinct intranuclear locations.

Two distinct intranuclear locations were identified for alternatively spliced RNA transcripts expressed from the pNL4-3 infectious molecular clone of human immunodeficiency virus (HIV) type 1. Multiply spliced HIV RNA encoding tat was detected within the nucleus in large clusters; immunostaining and colocalization studies using laser-scanning confocal microscopy revealed that these structures contained the non-small nuclear ribonucleoprotein RNA processing factor, SC35. In contrast, unspliced gag RNA was detected in much smaller granules distributed throughout the nucleus, with little or no association with SC35-containing granules. Analyses of nuclear RNA expressed from recombinant plasmids encoding gag (pCMVgag-2) alone or tat (pCMVtat-2) alone revealed distributions corresponding to those obtained with pNL4-3, indicating that expression within the context of the HIV provirus was not required for the distinct RNA locations detected for these transcripts. The presence of unspliced gag RNA in small granules was confirmed in infections of H9 T-lymphocytic cells, indicating that gag localization was not restricted to transient expression systems. The intranuclear distribution of gag RNA was dependent on specific RNA sequences. Deletion of a portion of the gag gene of pCMVgag-2, containing a cis-repressing inhibitory region, resulted in redirection of unspliced gag RNA from small granules into large SC35-containing clusters. The addition of the Rev-responsive element, RRE, to the deleted pCMVgag-2 construct resulted in RNA transcripts which were no longer associated with SC35. We also identified a cellular intron, rabbit beta-globin-intervening sequence 2 (IVS-2) which, when introduced into pCMVgag-2, redirected unspliced gag RNA into SC35-containing granules and permitted rev-independent Gag expression. These findings suggest that redirecting intranuclear RNA localization may influence gene expression. Color micrographs from this article are available for view at http//128.231.216.2/lmmhome.htm.

Alternative Splicing↗

PubMed1997

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.

Amino Acid Sequence↗

PubMed1997

A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA.

The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops. Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants. Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants. Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin. A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed.

Brassica↗