bluuebunny/crossref_metadata_2025 huggingface dataset:
The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells
Authors: Chang Y E , Van Sant C , Krug P W , Sears A E , Roizman B
Published: January 2020

Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.
Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome
Authors: Ito T , Lai M M
Published: January 2020

Hepatitis C virus (HCV) contains a positive-stranded RNA genome of approximately 9.5 kb. Despite the overall sequence diversity among individual HCV isolates, the 3'-end 98 nucleotides (nt) of the HCV RNA, which constitute part of the 3'-untranslated region (3'-UTR), are highly conserved. This conserved region may contain the cis-acting signals for RNA replication involving possibly both viral and cellular proteins. We carried out RNase digestion studies, which revealed that this 98-nt region contains three stem-loops but may also assume alternative structures. We further performed UV cross-linking experiments to detect cellular proteins that bound to this region. A 58-kDa cellular protein (p58) was detected. Its binding site was mapped to the stem-loops 2 and 3, which are the most conserved region of the 3'-UTR. Site-directed mutagenesis studies revealed that both stem structures and specific nucleotide sequence within the two loops are important for p58 binding. Mutations that disrupted stem structures abolished protein binding, while the compensatory mutations restored its binding. This region also contains partial sequence similarity to the reported consensus binding sequence for polypyrimidine tract-binding protein (PTB) (a 57-kDa protein). The UV-cross-linked protein could be immunoprecipitated with the anti-PTB antibody, and the recombinant PTB bound to the HCV 3'-UTR with the same binding specificity as p58, establishing that this protein is PTB. However, the reported PTB-binding sequence was not sufficient, but rather the entire stem-loops 2 and 3 were required, for PTB binding; thus, its binding specificity is significantly different from the reported PTB-binding sequence requirement. This protein was detected in both the nuclei and cytoplasm of most mammalian cell lines tested and human primary hepatocytes. PTB may participate in the regulation of HCV RNA synthesis or translation.
The N-terminal side of the origin-binding domain of simian virus 40 large T antigen is involved in A/T untwisting
Authors: Chen L , Joo W S , Bullock P A , Simmons D T
Published: January 2020

We investigated the role of the N-terminal side of simian virus 40 (SV40) large T antigen's origin-binding domain in the initiation of virus DNA replication by analyzing the biochemical activities of mutants containing single point substitutions or deletions in this region. Four mutants with substitutions at residues between 121 and 135 were partially defective in untwisting the A/T-rich track on the late side of the origin but were normal in melting the imperfect palindrome (IP) region on the early side. Deletion of the N-terminal 109 amino acids had no effect on either activity, whereas a longer deletion, up to residue 123, greatly reduced A/T untwisting but not IP melting. These results indicate that the region from residue 121 to 135 is important for A/T untwisting but not for IP melting and demonstrate that these activities are separable. Two point substitution mutants (126PS and 135PL) were characterized further by testing them for origin DNA binding, origin unwinding, oligomerization, and helicase activity. These two mutants were completely defective in origin (form U(R)) unwinding but normal in the other activities. Our results demonstrate that a failure to normally untwist the A/T track is correlated with a defect in origin unwinding. Further, they indicate that some mutants with substitutions in the region from residue 121 to 135 interact with origin DNA incorrectly, perhaps by failing to make appropriate contacts with the A/T-rich DNA.
Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity
Authors: Smelkova N V , Borowiec J A
Published: January 2020

Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.
A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA
Authors: Hellendoorn K , Verlaan P W , Pleij C W
Published: January 2020

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.
Functional complementation of UL3.5-negative pseudorabies virus by the bovine herpesvirus 1 UL3.5 homolog
Authors: Fuchs W , Granzow H , Mettenleiter T C
Published: January 2020

The UL3.5 gene is positionally conserved but highly variable in size and sequence in different members of the Alphaherpesvirinae and is absent from herpes simplex virus genomes. We have shown previously that the pseudorabies virus (PrV) UL3.5 gene encodes a nonstructural protein which is required for secondary envelopment of intracytoplasmic virus particles in the trans-Golgi region. In the absence of UL3.5 protein, naked nucleocapsids accumulate in the cytoplasm, release of infectious virions is drastically reduced, and plaque formation in cell culture is inhibited (W. Fuchs, B. G. Klupp, H. Granzow, H.-J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996). To assay functional complementation by a heterologous herpesviral UL3.5 protein, the UL3.5 gene of bovine herpesvirus 1 (BHV-1) was inserted at two different sites within the genome of UL3.5-negative PrV. In cells infected with the PrV recombinants the BHV-1 UL3.5 gene product was identified as a 17-kDa protein which was identical in size to the UL3.5 protein detected in BHV-1-infected cells. Expression of BHV-1 UL3.5 compensated for the lack of PrV UL3.5, resulting in a ca. 1,000-fold increase in virus titer and restoration of plaque formation in cell culture. Also, the intracellular block in viral egress was resolved by the BHV-1 UL3.5 gene. We conclude that the UL3.5 proteins of PrV and BHV-1 are functionally related and are involved in a common step in the egress of alphaherpesviruses.
Mutational analysis of human T-cell leukemia virus type 2 Tax
Authors: Ross T M , Minella A C , Fang Z Y , Pettiford S M , Green P L
Published: January 2020

A mutational analysis of human T-cell leukemia virus type 2 (HTLV-2) Tax (Tax-2) was performed to identify regions within Tax-2 important for activation of promoters through the CREB/ATF or NF-kappaB/Rel signaling pathway. Tax-2 mutations within the putative zinc-binding region as well as mutations at the carboxy terminus disrupted CREB/ATF transactivation. A single mutation within the central proline-rich region of Tax-2 disrupted the transactivation of the NF-kappaB/Rel pathway. Surprisingly, this mutation, which is thought to be in a separate activation domain, was suppressed by mutations within or around the putative zinc-binding region, suggesting an interaction between these two regions. These analyses indicate that the functional regions or domains important for transactivation through the CREB/ATF or NF-kappaB/Rel signaling pathway are similar, but not identical, in Tax-1 and Tax-2. Identification of these distinct Tax-2 mutants should facilitate comparative biological studies of HTLV-1 and HTLV-2 and ultimately lead to the determination of the functional importance of Tax trans-acting capacities in T-lymphocyte transformation by HTLV.
Implication of a cis-acting element in the cytoplasmic accumulation of unspliced Borna disease virus RNAs
Authors: Schneider P A , Schwemmle M , Lipkin W I
Published: January 2020

Borna disease virus (BDV), the prototype of a new family within the order Mononegavirales, is unusual in its nuclear localization for replication and transcription and use of RNA splicing for gene expression. The BDV antigenome contains three transcription units and six major open reading frames. Multicistronic RNAs containing two introns are elaborated from the third transcription unit. Differential splicing of the two introns and cytoplasmic accumulation of the unspliced and partially spliced RNA are critical for the balanced expression of the putative matrix protein, glycoprotein, and polymerase. To investigate the mechanisms for cytoplasmic expression of unspliced and partially spliced BDV transcripts, the levels of these transcripts were measured in the cytoplasm of infected COS-7 cells and noninfected COS-7 cells transfected with plasmids containing 2.8-kb cDNA inserts representing either wild-type or mutant BDV RNA from the third transcription unit. Analysis of truncation mutations allowed the identification of a cis-acting element present within the 3' end of the BDV 2.8-kb transcript that facilitated the cytoplasmic accumulation of unspliced BDV transcripts through nucleocytoplasmic transport. The nucleocytoplasmic transport activity was not dependent on the presence of BDV proteins. Gel-shift assays revealed that the cis-acting element binds specifically to host cytoplasmic and nuclear proteins.
Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus
Authors: Wingfield P T , Stahl S J , Thomsen D R , Homa F L , Booy F P , Trus B L , Steven A C
Published: January 2020

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.
Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease
Authors: Ro Y T , Scheffter S M , Patterson J L
Published: January 2020

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.
Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis
Authors: Doedens J R , Giddings T H , Kirkegaard K
Published: January 2020

Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.
Analysis of the internal replication sequence indicates that there are three elements required for efficient replication of minute virus of mice minigenomes
Authors: Brunstein J , Astell C R
Published: January 2020

Prior analysis of minigenomes of minute virus of mice carried out by our laboratory indicated that sequences within the region of nucleotides 4489 to 4695, inboard of the 5' palindrome, are required for efficient DNA replication of the virus and are the site of specific interactions with unidentified factors present in a host cell nuclear extract (P. Tam and C. R. Astell, Virology 193:812-824, 1993; P. Tam and C. R. Astell, J. Virology 68:2840-2848, 1994). In order to examine this region in finer detail, a comprehensive library of linker-scanning mutants spanning the region was tested for the ability to support replication of minigenome constructs and for the ability to interact with host cell factors. Three short discrete sequence elements critical for replication competence were observed. Binding of host cell nuclear factors was localized to four sites, with two major complexes each appearing to have two binding sites within the region. All factor binding sites were found to be directly adjacent to or overlapping with sequence elements contributing to replication competence, and evidence suggesting a correlation between factor binding and minigenome replication is presented. A possible model is proposed for function of a viral origin within the region of the internal replication sequence which addresses the still-unresolved problem of how parvoviruses overcome the thermodynamic energy barrier involved in the rearrangement of the 5'-terminal palindrome from an extended form to a hairpin conformation.
Fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase
Authors: Ding J W , Ning Q , Liu M F , Lai A , Leibowitz J , Peltekian K M , Cole E H , Fung L S , Holloway C , Marsden P A , Yeger H , Phillips M J , Levy G A
Published: January 2020

Activation of the immune coagulation system has been implicated in the pathogenesis of fulminant liver failure caused by murine hepatitis virus strain 3 (MHV-3). The recent discovery of the fgl2 gene, which encodes for MHV-3-induced prothrombinase (fgl2 prothrombinase), allows for fundamental studies to determine the molecular basis for fulminant liver failure. Transcription of the fgl2 gene and translation of the protein it encodes were examined in the liver and other organs of susceptible mice following MHV-3 infection. No constitutive expression of the fgl2 gene or the fgl2 prothrombinase was detected. Within 12 to 24 h of MHV-3 infection, however, fgl2 gene transcripts were detected in large amounts in the liver, spleen, and lungs, all of which are rich in reticuloendothelial cells, but were only focally present in small amounts in the kidney and brain. There was sequential detection of fgl2 prothrombinase in the liver, where it was localized specifically to the endothelium of intrahepatic veins and hepatic sinusoids; this was allowed by fibrin deposition, which resulted in confluent hepatocellular necrosis. These results provide further evidence for the role of the selective expression of this novel fgl2 prothrombinase in the pathogenesis of MHV-3-induced fulminant liver failure.
The vaccinia virus I1 protein is essential for the assembly of mature virions
Authors: Klemperer N , Ward J , Evans E , Traktman P
Published: January 2020

The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.
Direct demonstration of retroviral recombination in a rhesus monkey
Authors: Wooley D P , Smith R A , Czajak S , Desrosiers R C
Published: January 2020

Recombination may be an important mechanism for increasing variation in retroviral populations. Retroviral recombination has been demonstrated in tissue culture systems by artificially creating doubly infected cells. Evidence for retroviral recombination in vivo is indirect and is based principally on the identification of apparently mosaic human immunodeficiency virus type 1 genomes from phylogenetic analyses of viral sequences. We infected a rhesus monkey with two different molecularly cloned strains of simian immunodeficiency virus. One strain of virus had a deletion in vpx and vpr, and the other strain had a deletion in nef. Each strain on its own induced low virus loads and was nonpathogenic in rhesus monkeys. When injected simultaneously into separate legs of the same monkey, persistent high virus loads and declines in CD4+ lymphocyte concentrations were observed. Analysis of proviral DNA isolated directly from peripheral blood mononuclear cells showed that full-length, nondeleted SIVmac239 predominated by 2 weeks after infection. These results provide direct experimental evidence for genetic recombination between two different retroviral strains in an infected host. The results illustrate the ease and rapidity with which recombination can occur in an infected animal and the selection that can occur for variants generated by genetic recombination.
A major human immunodeficiency virus type 1-initiated killing pathway distinct from apoptosis
Authors: Kolesnitchenko V , King L , Riva A , Tani Y , Korsmeyer S J , Cohen D I
Published: January 2020

We have investigated the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with T-cell-tropic, cytopathic human immunodeficiency virus type 1 (HIV-1), by employing diverse strategies to inhibit PCD or to detect its common end-stage sequelae. When Bcl-2-transfected cell lines were infected with HIV-1, their viability was only slightly higher than that of control infections. Although the adenovirus E1B 19-kDa protein has been reported to be a stronger competitor of apoptosis than Bcl-2, it did not inhibit HIV-mediated cell death better than Bcl-2 protein. Competition for Fas ligand or inactivation of the Fas pathway secondary to intracellular mutation (MOLT-4 T cells) also had modest effects on overall cell death during acute HIV infection. In contrast to these observations with HIV infection or with HIV envelope-initiated cell death, Tat-expressing cell lines were much more susceptible (200% enhancement) to Fas-induced apoptosis than controls and Bcl-2 overexpression strongly (75%) inhibited this apoptotic T-cell death. PCD associated with FasR ligation resulted in the cleavage of common interleukin-1beta-converting enzyme (ICE)-protease targets, poly(ADP-ribose) polymerase (PARP) and pro-ICE, whereas cleaved products were not readily detected during HIV infection of peripheral blood mononuclear cells or T-cell lines even during periods of extensive cell death. These results indicate that one important form of HIV-mediated cell killing proceeds by a pathway that lacks the characteristics of T-cell apoptosis. Our observations support the conclusion that at least two HIV genes (env and tat) can kill T cells by distinct pathways and that an envelope-initiated process of T-cell death can be discriminated from apoptosis by many of the properties most closely associated with apoptotic cell death.
A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus
Authors: Desrosiers R C , Sasseville V G , Czajak S C , Zhang X , Mansfield K G , Kaur A , Johnson R P , Lackner A A , Jung J U
Published: January 2020

A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus.
The feline leukemia virus long terminal repeat contains a potent genetic determinant of T-cell lymphomagenicity
Authors: Pantginis J , Beaty R M , Levy L S , Lenz J
Published: January 2020

Feline leukemia virus (FeLV) is an important pathogen of domestic cats. The most common type of malignancy associated with FeLV is T-cell lymphoma. SL3-3 (SL3) is a potent T-cell lymphomagenic murine leukemia virus. Transcriptional enhancer sequences within the long terminal repeats (LTRs) of SL3 and other murine retroviruses are crucial genetic determinants of the pathogenicities of these viruses. The LTR enhancer sequences of FeLV contain identical binding sites for some of the transcription factors that are known to affect the lymphomagenicity of SL3. To test whether the FeLV LTR contains a genetic determinant of lymphomagenicity, a recombinant virus that contained the U3 region of a naturally occurring FeLV isolate, LC-FeLV, linked to the remainder of the genome of SL3 was generated. When inoculated into mice, the recombinant virus induced T-cell lymphomas nearly as quickly as SL3. Moreover, the U3 sequences of LC-FeLV were found to have about half as much transcriptional activity in T lymphocytes as the corresponding sequences of SL3. This level of activity was severalfold higher than that of the LTR of weakly leukemogenic Akv virus. Thus, the FeLV LTR contains a potent genetic determinant of T-cell lymphomagenicity. Presumably, it is adapted to be recognized by transcription factors present in T cells of cats, and this yields a relatively high level of transcription that allows the enhancer to drive the requisite steps in the process of lymphomagenesis.
Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein
Authors: Cao J , Sullivan N , Desjardin E , Parolin C , Robinson J , Wyatt R , Sodroski J
Published: January 2020

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.
The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells
Authors: Hallenberger S , Moulard M , Sordel M , Klenk H D , Garten W
Published: January 2020

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.
Nuclear targeting of incoming human foamy virus Gag proteins involves a centriolar step
Authors: Saïb A , Puvion-Dutilleul F , Schmid M , Périès J , de Thé H
Published: January 2020

The pathways used in the transport of retroviral genomes to the nucleus are poorly identified. Analyzing the intracellular localization of incoming foamy viruses, we have found that the Gag antigens and the viral genome accumulate in a distinct perinuclear domain identified as the centrosome. Colchicine treatment completely abolished pericentriolar targeting of human foamy virus (HFV) proteins, suggesting a role for microtubules in the transport of the incoming viral proteins to the centrioles. Finally, we demonstrate that, similarly to human immunodeficiency virus DNA, HFV DNA can enter the nucleus of G1/S-phase-arrested cells, although no viral gene expression can be observed. Recent observations have demonstrated that foamy viruses have several features not shared by other retroviruses. The intracellular route of the incoming Gag antigens may constitute a new specificity of this class of viruses.
Immunocytochemical analysis of Uukuniemi virus budding compartments: role of the intermediate compartment and the Golgi stack in virus maturation
Authors: Jäntti J , Hildén P , Rönkä H , Mäkiranta V , Keränen S , Kuismanen E
Published: January 2020

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.
Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells
Authors: Meier J L , Stinski M F
Published: January 2020

Differentiation-dependent expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 and IE2, may partly govern virus replication in monocytic THP-1 and embryonal carcinoma (Tera-2) cells. The modulator of the MIE promoter was shown previously in transient transfection assays to repress transcription from promoter segments in undifferentiated THP-1 and Tera-2 cells but not in differentiated cells. To determine the biological importance of these findings, we constructed a recombinant HCMV (r delta MSVgpt) without a modulator. In comparison to wild-type (WT) virus, r delta MSVgpt exhibits a slight delay in growth in human fibroblasts, but there is no appreciable change in IE1 and IE2 transcription. Moreover, there is no appreciable change in the early/late kinetics of transcription of RNAs colinear with the predicted UL128 coding region, which is adjacent to the modulator, although the size distribution and abundance of these RNAs are altered. In infected undifferentiated THP-1 and Tera-2 cells, WT and r alpha MSVgpt viruses produce minimal but comparable amounts of IE1 RNAs. The genomes of both viruses are detectable in similar amounts within these undifferentiated cells. Induction of cellular differentiation before infection overcomes the block in MIE gene transcription. WT and r alpha MSVgpt infections of differentiated THP-1 cells produce similar levels of IE1 and IE2 RNAs. Thus, differentiation-dependent control of MIE gene transcription involves regulatory mechanisms other than the modulator. Possible alternative functions of the modulator are discussed.
Rescue of rinderpest virus from cloned cDNA
Authors: Baron M D , Barrett T
Published: January 2020

Rinderpest virus is a morbillivirus and is the causative agent of a widespread and important disease of cattle. The viral genome is a single strand of RNA in the negative sense. We have constructed plasmids containing cDNA copies of the 5' and 3' termini of the virus separated by a reporter gene and have shown that antigenome-sense RNA transcripts of these model genomes can be replicated, transcribed, and packaged by helper virus, both rinderpest virus and the related measles virus. Further, these genome analogs can be replicated and transcribed by viral proteins expressed from cDNA clones by using a recombinant vaccinia virus expressing T7 RNA polymerase (MVA-T7). Using this latter system, we have rescued live rinderpest virus from a full-length cDNA copy of the genome of the RBOK vaccine strain. The recombinant virus appears to grow in tissue culture identically to the original virus.
Assembly of herpes simplex virus capsids using the human cytomegalovirus scaffold protein: critical role of the C terminus
Authors: Oien N L , Thomsen D R , Wathen M W , Newcomb W W , Brown J C , Homa F L
Published: January 2020

An essential step in assembly of herpes simplex virus (HSV) type 1 capsids involves interaction of the major capsid protein (VP5) with the C terminus of the scaffolding protein (encoded by the UL26.5 gene). The final 12 residues of the HSV scaffolding protein contains an A-X-X-F-V/A-X-Q-M-M-X-X-R motif which is conserved between scaffolding proteins found in other alphaherpesviruses but not in members of the beta- or gamma-herpesviruses. Previous studies have shown that the bovine herpesvirus 1 (alphaherpesvirus) UL26.5 homolog will functionally substitute for the HSV UL26.5 gene (E. J. Haanes et al., J. Virol. 69:7375-7379, 1995). The homolog of the UL26.5 gene in the human cytomegalovirus (HCMV) genome is the UL80.5 gene. In these studies, we tested whether the HCMV UL80.5 gene would substitute for the HSV UL26.5 gene in a baculovirus capsid assembly system that we have previously described (D. R. Thomsen et al., J. Virol. 68:2442-2457, 1994). The results demonstrate that (i) no intact capsids were assembled when the full-length or a truncated (missing the C-terminal 65 amino acids) UL80.5 protein was tested; (ii) when the C-terminal 65 amino acids of the UL80.5 protein were replaced with the C-terminal 25 amino acids of the UL26.5 protein, intact capsids were made and direct interaction of the UL80.5 protein with VP5 was detected; (iii) assembly of intact capsids was demonstrated when the sequence of the last 12 amino acids of the UL80.5 protein was changed from RRIFVA ALNKLE to RRIFVAAMMKLE; (iv) self-interaction of the scaffold proteins is mediated by sequences N terminal to the maturation cleavage site; and (v) the UL26.5 and UL80.5 proteins will not coassemble into scaffold structures. The results suggest that the UL26.5 and UL80.5 proteins form a scaffold by self-interaction via sequences in the N termini of the proteins and emphasize the importance of the C terminus for interaction of scaffold with the proteins that form the capsid shell.
Mother-to-infant transmission of human immunodeficiency virus type 1 involving five envelope sequence subtypes
Authors: Contag C H , Ehrnst A , Duda J , Bohlin A B , Lindgren S , Learn G H , Mullins J I
Published: January 2020

Genetic analysis of human immunodeficiency virus type 1 (HIV-1) from cases of mother-to-infant transmission were analyzed in an effort to provide insights into the viral selection that may occur during transmission, as well as the timing and source of transmitted viruses. HIV-1 env genes obtained from seven mothers and their perinatally infected infants in Sweden were studied. Five envelope sequence clades (A to E) were found to be represented. We used a heteroduplex tracking assay (HTA) to assess the genetic relatedness between early viral isolates from the infants and serial maternal virus populations taken during pregnancy and at delivery. HTA findings were used to select for DNA sequence analysis maternal virus populations that were either closely or more distantly related to the infant virus. In each case, nucleotide sequence analysis confirmed the genetic relationships inferred by the HTA. Only maternal peripheral blood was sampled, and large sets of maternal specimens throughout pregnancy were generally not available. However, no consistent correlation was found to support the hypothesis that infant viruses should match blood-derived maternal virus genotypes found early in pregnancy if infants were found to be infected at birth or, conversely, that infant viruses should match blood-derived maternal virus genotypes found at delivery if infants were found to be infected only some time later.
The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA
Authors: Perales B , Ortín J
Published: January 2020

The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA.
The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein
Authors: Donald R G , Lawrence D M , Jackson A O
Published: January 2020

The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.
Functions of the tobacco etch virus RNA polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa)
Authors: Li X H , Valdez P , Olvera R E , Carrington J C
Published: January 2020

The NIb protein of tobacco etch potyvirus (TEV) possesses several functions, including RNA-dependent RNA polymerase and nuclear translocation activities. Using a reporter protein fusion strategy, NIb was shown to contain two independent nuclear localization signals (NLS I and NLS II). NLS I was mapped to a sequence within amino acid residues 1 to 17, and NLS II was identified between residues 292 and 316. Clustered point mutations resulting in substitutions of basic residues within the NLSs were shown previously to disrupt nuclear translocation activity. These mutations also abolished TEV RNA amplification when introduced into the viral genome. The amplification defects caused by each NLS mutation were complemented in trans within transgenic cells expressing functional NIb, although the level of complementation detected for each mutant differed significantly. Combined with previous results (X. H. Li and J. C. Carrington, Proc. Natl. Acad. Sci. USA 92:457-461, 1995), these data suggest that the NLSs overlap with essential regions necessary for NIb trans-active function(s). The fact that NIb functions in trans implies that it must interact with one or more other components of the genome replication apparatus. A yeast two-hybrid system was used to investigate physical interactions between NIb and several other TEV replication proteins, including the multifunctional VPg/proteinase NIa and the RNA helicase CI. A specific interaction was detected between NIa and NIb. Deletion of any of five regions spanning the NIb sequence resulted in NIb variants that were unable to interact with NIa. Clustered point mutations affecting the conserved GDD motif or NLS II within the central region of NIb, but not mutations affecting NLS I near the N terminus, reduced or eliminated the interaction. The C-terminal proteinase (Pro) domain of NIa, but not the N-terminal VPg domain, interacted with NIb. The effects of NIb mutations within NLS I, NLS II, and the GDD motif on the interaction between the Pro domain and NIb were identical to the effects of these mutations on the interaction between full-length NIa and NIb. These data are compatible with a model in which NIb is directed to replication complexes through an interaction with the Pro domain of NIa.
Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors
Authors: Chen D S , Asanaka M , Chen F S , Shively J E , Lai M M
Published: January 2020

Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.