Efficient Viral Growth Research Articles

Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HΔgK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HΔgK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early–late kinetics, and multiple forms of the protein exhibiting M rs between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.

653. Activation of an ATM/chk2-Dependent Pathway Is Required for HSV-1 Protein ICp0 Induced G2/M Cell Cycle Arrest

Herpes simplex virus type 1 (HSV-1) replication involves the expression of its genes in a interdependent and well ordered cascade. The immediate early (IE) genes are expressed first on viral DNA entry into the nucleus and do not require expression of any other viral functions. The IE gene product, ICP0, plays an important role in enhancing early and late gene expression and is required for virus reactivation from latency in peripheral nerves. ICP0 is also toxic for dividing cells primarily through its ability to induce cell cycle arrest at G1 and S in synchronized cells and G2/M arrest in nonsynchronized cells ( ). The mechanism of cell cycle arrest is unknown. In this report, we confirm that mutant viruses that only express ICP0 cause G2/M arrest. Following infection, ICP0 induces ATM phosphorylation at tyrosine 1981 and CHK2 phosphorylation at tyrosine 68, P53 phosphorylated in serine 15 and 20. These events lead to G2 arrest. Chk2-/- cells can not cause G2/M arrest after virus infection but rather results in apoptosis. Together these results provide a mechanism through which ICP0 arrests cell division an event important to efficient virus growth.

The influenza B virus nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction.

We analyzed the functions of the influenza B virus nonstructural NS1-B protein, both by utilizing a constructed mutant virus (Delta NS1-B) lacking the NS1 gene and by testing the activities of the protein when expressed in cells. The mutant virus replicated to intermediate levels in 6-day-old embryonated chicken eggs that contain an immature interferon (IFN) system, whereas older eggs did not support viral propagation to a significant extent. The Delta NS1-B virus was a substantially stronger inducer of beta IFN (IFN-beta) transcripts in human lung epithelial cells than the wild type, and furthermore, transiently expressed NS1-B protein efficiently inhibited virus-dependent activation of the IFN-beta promoter. Interestingly, replication of the Delta NS1-B knockout virus was attenuated by more than 4 orders of magnitude in tissue culture cells containing or lacking functional IFN-alpha/beta genes. These findings show that the NS1-B protein functions as a viral IFN antagonist and indicate a further requirement of this protein for efficient viral replication that is unrelated to blocking IFN effects.

Changes in rhinovirus protein 2C allow efficient replication in mouse cells.

Rhinovirus type 16 was found to replicate in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However, infection of these cells at a low multiplicity of infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1 L cells. Infection of mouse cells with 16/L leads to higher virus titers, increased production of RNA, and total cytopathic effect. Three amino acid changes were identified in the P2 region of virus 16/L, and the adaptation phenotype mapped to two changes in protein 2C. The characterization of a rhinovirus host range mutant will facilitate the investigation of cellular proteins required for efficient viral growth and the development of a murine model for rhinovirus infection.

Mapping of functional regions in the amino-terminal portion of the herpes simplex virus ICP27 regulatory protein: importance of the leucine-rich nuclear export signal and RGG Box RNA-binding domain.

Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.

Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: relationship to growth in the duck intestine.

The 1957 human pandemic strain of influenza A virus contained an avian virus hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for the human receptor, N-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an alpha2-6 bond (NeuAcalpha2-6Gal). Although the NA retained considerable specificity for NeuAcalpha2-3Gal, its original substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift in substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient viral growth in the intestines of orally inoculated ducks. The growth-supporting capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, we localized the determinants of high specific NA activity to a region containing six amino acid substitutions in A/England/12/62: Ser331-->Arg, Asp339-->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and Pro431-->Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs for their cellular receptor.

Bovine Herpesvirus 1 Glycoprotein G Is Required for Prevention of Apoptosis and Efficient Viral Growth in Rabbit Kidney Cells

In rabbit kidney (RK13) cells, gG-negative BHV-1 exhibited significant defects in plaque formation and growth compared to that of gG-positive BHV-1. RK13 cells infected with gG-negative BHV-1 exhibited a distinctive CPE and contained a larger number of cells stained with trypan blue dye compared to those infected with gG-positive strains, suggesting that gG-negative BHV-1 inflicted more damage to the infected cells than gG-positive BHV-1. Apoptotic cell death was induced in RK13 cells infected with gG-negative BHV-1 within 8 h. In contrast, the onset of apoptosis in gG-positive BHV-1-infected RK13 cells was around 12–16 h postinfection. In the presence of caspase inhibitor Z-Asp-CH2-DCB, multiplication of gG-negative minus BHV-1 was significantly increased. These results demonstrate that BHV-1 gG is involved in stabilizing the cell structure, postponing apoptotic process, and efficient BHV-1 replication in infected RK13 cells.

Role of the C-terminal tryptophan residue for the structure-function of the alphavirus capsid protein

The Semliki Forest virus capsid protein is a multifunctional protein which packages genomic RNA into nucleocapsid structures and binds to viral spike protein during budding. In addition, the capsid protein has an autoproteolytic activity whereby the C-terminal tryptophan is used as the substrate for cotranslational cleavage of the viral structure polyprotein. The autoproteolytic domain of the capsid protein has a chymotrypsin-like fold but has two additional short β-strands which place the tryptophan into the active site. Here, we have substituted the C-terminal tryptophan of Semliki Forest virus capsid protein for alanine, arginine and phenylalanine and analysed the effects on different functions of the C protein such as nucleocapsid formation, spike binding and autoproteolytic activity. We found that (i) tryptophan is a better substrate for the autoproteolytic activity, (ii) the wild-type tryptophan is the only residue that allows efficient viral growth and (iii) an aromatic residue is important for correct initial folding and stability of the protein.

HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import.

Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.

Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in Vitro and are important for virus growth in Vivo

The adenovirus inverted terminal repeat (ITR) contains a number of cis-acting elements that are involved in the initiation of viral DNA replication, as well as multiple binding motifs for the cellular transcription factors SP1 and ATF. In this study, we utilized a Hela cell transcription extract to demonstrate that the adenovirus type 5 ITR promotes bidirectional transcription in vitro. Primer extension analyses demonstrated that the ITR directed transcription at initiation sites both within the terminal repeat and at fixed distances outside of the ITR. The ITR also strongly stimulated transcription at the early region 1A (E1A) initiation site when it was situated immediately upstream of the E1A TATA ☐ region. Deletion and point mutational analyses demonstrated that two distinct cis-acting elements were involved in these ITR-dependent transcriptional activities in vitro. Cellular transcription factors SP1 and ATF were previously shown to bind to these two regions. Analysis of viral mutants in vivo demonstrated that the NFIII/OCT-1 binding site and a conserved ATF motif were important for efficient viral growth. Regulatory elements in the ITR flanking region were found to functionally substitute for these sites.

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.

The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner

Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K.

A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame

Varicella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmw175 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV-1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmw175 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmw175 functions, D30EBA is able to grow only in cell lines which express Vmw175 constitutively. The resulting recombinant virus. HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmw175 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.

Pseudorabies virus glycoprotein gIII is required for efficient virus growth in tissue culture.

Glycoprotein gIII of pseudorabies virus is a major antigen found in the envelopes of virus particles as well as in and on the surfaces of infected cells. It is not an essential gene product for virus growth in tissue culture. In this report, we provide evidence that, although it is not essential, the gIII protein is required for efficient virus growth and that gIII mutants are quickly outgrown by wild-type virus in mixed infections.

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.