Early Steps Of Viral Infection Research Articles

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies.

Ultrastructural Damages to H1N1 Influenza Virus Caused by Vapor Essential Oils.

Influenza viruses are transmitted from human to human via airborne droplets and can be transferred through contaminated environmental surfaces. Some works have demonstrated the efficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined the liquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viral cytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs and their vapor phase by the head-space gas chromatography–mass spectrometry technique, observing that the major component found in each liquid EO is the same one of the corresponding vapor phases, with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrity of virus particles was checked by negative staining transmission electron microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes. We speculated that the most abundant components of the vapor EOs might directly interfere with influenza virus envelope structures or mask viral structures important for early steps of viral infection.

Hamster organotypic modeling of SARS-CoV-2 lung and brainstem infection

SARS-CoV-2 has caused a global pandemic of COVID-19 since its emergence in December 2019. The infection causes a severe acute respiratory syndrome and may also spread to central nervous system leading to neurological sequelae. We have developed and characterized two new organotypic cultures from hamster brainstem and lung tissues that offer a unique opportunity to study the early steps of viral infection and screening antivirals. These models are not dedicated to investigate how the virus reaches the brain. However, they allow validating the early tropism of the virus in the lungs and demonstrating that SARS-CoV-2 could infect the brainstem and the cerebellum, mainly by targeting granular neurons. Viral infection induces specific interferon and innate immune responses with patterns specific to each organ, along with cell death by apoptosis, necroptosis, and pyroptosis. Overall, our data illustrate the potential of rapid modeling of complex tissue-level interactions during infection by a newly emerged virus.

Study on suitable analysis method for HIV-1 non-catalytic integrase inhibitor

BackgroundIntegrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs.MethodsIn this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3′-processing inhibition assay were performed.ResultsDuring screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication.ConclusionAccordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

Bovine Lactoferrin Prevents Influenza A Virus Infection by Interfering with the Fusogenic Function of Viral Hemagglutinin.

Bovine lactoferrin (bLf) is an iron-binding glycoprotein folded in two symmetric globular lobes (N- and C-lobes) with potent antimicrobial and immunomodulatory activities. Recently, we have shown that bLf, and in particular its C-lobe, interacts with influenza A virus hemagglutinin and prevents infection by different H1 and H3 viral subtypes. Influenza virus hemagglutinin (HA), and in particular its highly conserved fusion peptide involved in the low-pH-mediated fusion process, plays a significant role in the early steps of viral infection and represents an attractive target for the development of anti-influenza drugs. In the present research, we further investigated the influence of low pH on the interactions between bLf and influenza A H1N1 virus by different techniques, such as enzyme-linked immunosorbent assay, electron microscopy, hemolysis inhibition assay, and time course assay. Our results demonstrate that lactoferrin interaction with influenza hemagglutinin at low pH induces alterations that stabilize the conformation of the hemagglutinin, resulting in the inhibition of the fusion peptide activity. Taken together, our data allowed to better characterize the HA-specific inhibiting activity of bLf and to confirm HA as a good target for drug development.

Prokaryotic expression and identification of scavenger receptor B2.

There is still no effective clinical antiviral drug against human enterovirus 71 (EV71) infection, which causes hand, foot and mouth disease (HFMD) in children. Scavenger receptor class B member 2 (SCARB2) is an important receptor of EV71 as it plays a vital role in the early steps of viral infection. In this study, recombinant SCARB2 protein was expressed and purified in a prokaryotic expression system, and was identified by western blot with a monoclonal antibody and mass spectrometry analysis. Detection of the sera from mice immunized with the recombinant SCARB2 protein using ELISA and western blot showed good immunogenicity of the recombinant protein. Furthermore, in the neutralization test cytopathic effect was significantly decreased when EV71 was incubated with the immune sera before infection. In summary, the SCARB2 protein was expressed successfully, and the immune sera showed obvious antiviral effect against EV71. This study provides useful information about the interaction mechanism between SCARB2 and EV71, and is also helpful for further clinical treatment research of HFMD.

Comparative infection of autologous primary T cells and monocytes derived DCs using cell-free virus preparation, viral biofilm, pseudotyped-virus or viral synapse

HTLV-1 infected T cells and blood dentritic cells (DCs) can be found in HTLV-1 carriers. It is currently believed that T cell infection requires cell-cell contact, while DCs could also be infected with cell-free virus. However, a comparative study using different modes of viral preparation for infecting human primary autologous T and DC cells has not been performed. Supernatant from HTLV-1 chronically infected T cells, cell conjugates between HTLV-1 infected cells and target cells, or purified viral biofilm were used side-by-side to infect autologous human primary lymphocytes and monocytes-derived dendritic cells. To monitor the early steps of viral infection, we also used pseudotyped viruses carrying either the HTLV-1 or the VSV envelopes. Infection was then followed by flow cytometry, ELISA, immunofluorescence, real-time PCR and alu-PCR. Our first data show that, depending on the source of viruses, lymphocytes have distinct susceptibility to HTLV-1 infection. The impact of the viral source in the spread of HTLV-1 infection will be discussed.

Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC50’s were in the 50μM range with no toxicity to the cells at concentrations up to 200μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

The Cytoplasmic Domain of Marburg Virus GP Modulates Early Steps of Viral Infection

Marburg virus infection is mediated by the only viral surface protein, GP, a trimeric type I transmembrane protein. While its ectodomain mediates receptor binding and fusion of viral and cellular membranes and its transmembrane domain is essential for the recruitment of GP into budding particles by the matrix protein VP40, the role of the short cytoplasmic domain has remained enigmatic. Here we show that a missing cytoplasmic domain did not impair trimerization, intracellular transport, or incorporation of GP into infectious Marburg virus-like particles (iVLPs) but altered the glycosylation pattern as well as the recognition of GP by neutralizing antibodies. These results suggest that subtle conformational changes took place in the ectodomain. To investigate the function of the cytoplasmic domain during viral entry, a novel entry assay was established to monitor the uptake of filamentous VLPs by measuring the occurrence of luciferase-labeled viral nucleocapsids in the cytosol of target cells. This quantitative assay showed that the entry process of VLPs incorporating GP missing its cytoplasmic domain (GPΔCD) was impaired. Supporting these results, iVLPs incorporating a mutant GP missing its cytoplasmic domain were significantly less infectious than iVLPs containing wild-type GP. Taken together, the data indicate that the absence of the short cytoplasmic domain of Marburg virus GP may induce conformational changes in the ectodomain which impact the filoviral entry process.

Perspective of using the recombinant DNA-technology to control the spread of the African swine fever

The causative agent of ASF is a large deoxyvirus that has been assigned to Asfarviridae family. Previous studies have shown that ASFV p54 and p30 are essential viral proteins involved in the early steps of viral infection, whereas the adsorption of ASFV to susceptible cells is mediated by the structural virus protein p12, located within the outer envelope of the virion. It is believed that the lack of efficient ASFV vaccines might be due to unique molecular and biological properties of ASFV proteins responsible for virus–cell interactions: p30, p54 and p12. In this report, we describe the effect of in vitro blockage of ASF virus–cell interactions mediated by p54, p30 and p12. Computer analysis of p54 gene sequences of different ASFV field strains form GenBank was performed. According to this analysis ASFV viruses were distributed in several groups matching their serotype classification developed at our institute earlier. Immunization of rabbits with recombinant p54, p12 or p30 proteins induced antibodies which inhibited virus attachment or internalization. The serum of rabbits immunized with p54 of Magadi strain prevented virus attachment of Magadi group viruses only. In contrast, the serum from rabbits immunized with p30 (Magadi strain) rendered an in vitro inhibition of different ASFV groups replication.

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.

Glycoside analogs of β-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry

The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb 3 is known to facilitate HIV binding to both CD4 + as well as CD4 − cells. In an effort to develop small molecule HIV-1 entry inhibitors with improved solubility and efficacy, we have synthesized a series of C-glycoside analogs of GalCer and tested their anti HIV-1 activity. The analogs were tested for gp120 binding using a HIV-1 (IIIB) V3-loop specific peptide. Two of the six analogs that interfered with gp120 binding also inhibited HIV Env-mediated cell-to-cell fusion and viral entry in the absence of any significant cytotoxicity. Analogs with two side chains did not show inhibition of fusion and/or infection under identical conditions. The inhibition of virus infection seen by these compounds was not coreceptor dependent, as they inhibited CXCR4, CCR5 as well as dual tropic viruses. These compounds showed inhibition of HIV entry at early steps in viral infection since the compounds were inactive if added post viral entry. Temperature-arrested state experiments showed that the compounds act at the level of virus attachment to the cells likely at a pre-CD4 engagement step. These compounds also showed inhibition of VSV glycoprotein-pseudotyped virus. The results presented here show that the glycoside derivatives of GalCer with simple side chains may serve as a novel class of small molecule HIV-1 entry inhibitors that would be active against a number of HIV isolates as well as other enveloped viruses.

Characterization of the Early Steps of Infection of Primary Blood Monocytes by Human Immunodeficiency Virus Type 1

Blood-circulating monocytes migrate in tissues in response to danger stimuli and differentiate there into two major actors of the immune system: macrophages and dendritic cells. Given their migratory behavior and their pivotal role in the orchestration of immune responses, it is not surprising that cells of the monocyte lineage are the target of several viruses, including human immunodeficiency virus type 1 (HIV-1). HIV-1 replicates in monocytoid cells to an extent that is influenced by their differentiation status and modulated by exogenous stimulations. Unstimulated monocytes display a relative resistance to HIV infection mostly exerted during the early steps of the viral life cycle. Despite intensive studies, the identity of the affected step remains controversial, although it is generally assumed to take place after viral entry. We reexamine here the early steps of viral infection of unstimulated monocytes using vesicular stomatitis virus G protein-pseudotyped HIV-1 virions. Our data indicate that a first block to the early steps of infection of monocytes with these particles occurs at the level of viral entry. After entry, reverse transcription and integration proceed with extremely slow kinetics rather than being blocked. Once completed, viral DNA molecules delay entry into the nucleus and integration for up to 5 to 6 days. The inefficacy of these steps accounts for the resistance of monocytes to HIV-1 during the early steps of infection.

Structure of the Ebola Fusion Peptide in a Membrane-mimetic Environment and the Interaction with Lipid Rafts

The fusion peptide EBO(16) (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and (1)H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO(16) undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3(10)-helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO(16) can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.

SIVSM/HIV-2 Vpx proteins promote retroviral escape from a proteasome-dependent restriction pathway present in human dendritic cells

BackgroundVpx is a non-structural protein coded by members of the SIVSM/HIV-2 lineage that is believed to have originated by duplication of the common vpr gene present in primate lentiviruses. Vpx is incorporated into virion particles and is thus present during the early steps of viral infection, where it is thought to drive nuclear import of viral nucleoprotein complexes. We have previously shown that Vpx is required for SIVMAC-derived lentiviral vectors (LVs) infection of human monocyte-derived dendritic cells (DCs). However, since the requirement for Vpx is specific for DCs and not for other non-dividing cell types, this suggests that Vpx may play a role other than nuclear import.ResultsHere, we show that the function of Vpx in the infection of DCs is conserved exclusively within the SIVSM/HIV-2 lineage. At a molecular level, Vpx acts by promoting the accumulation of full length viral DNA. Furthermore, when supplied in target cells prior to infection, Vpx exerts a similar effect following infection of DCs with retroviruses as divergent as primate and feline lentiviruses and gammaretroviruses. Lastly, the effect of Vpx overlaps with that of the proteasome inhibitor MG132 in DCs.ConclusionOverall, our results support the notion that Vpx modifies the intracellular milieu of target DCs to facilitate lentiviral infection. The data suggest that this is achieved by promoting viral escape from a proteasome-dependent pathway especially detrimental to viral infection in DCs.

Macrophage Inflammatory Protein 1α Inhibits Postentry Steps of Human Immunodeficiency Virus Type 1 Infection via Suppression of Intracellular Cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.

Opening of Size-Selective Pores in Endosomes during Human Rhinovirus Serotype 2 In Vivo Uncoating Monitored by Single-Organelle Flow Analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.

Lactoferrin inhibits human papillomavirus binding and uptake in vitro

Lactoferrin (LF), a member of the transferrin family, is a bi-globular protein secreted in milk, saliva, tears, seminal fluid, endocervix and vaginal secretions. LF is an important player in the defence against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus and poliovirus. The antiviral activity of LF is directed against the early steps of viral infection and the LF antiviral effect against herpesvirus is mediated through LF binding to the herpesvirus receptor heparan sulfate. Human papillomavirus (HPV) causes genital warts and is a prerequisite for cervical cancer. HPV can also use heparan sulfate on the cell surface as a receptor. We studied the inhibition by LF on HPV entry by incubating HaCaT cells and HPV-16 virus-like particles (VLPs) with either human (HLF) or bovine lactoferrin (BLF). LF inhibited internalization of HPV-16 particles using CFDA-SE-labelled VLPs that only fluoresce after internalisation. By using a western blot assay we also found dose-dependent LF inhibition of HPV-16 VLP binding to the HaCaT cell surface. BLF was a more potent inhibitor of HPV entry than human LF. It was also clear that LF acted early in the HPV uptake process.

Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA.

Dengue fever is a mosquito-borne viral disease of global importance with no available antiviral therapy. We assessed the ability of mycophenolic acid (MPA), a drug currently used as an immunosuppressive agent, to inhibit dengue virus (DV) antigen expression, RNA replication, and virus production. Pharmacological concentrations of MPA effectively blocked DV infection, decreasing the percentage of infected cells by 99% and the levels of secreted virus by up to a millionfold. Results were reproduced with four hepatoma cell lines and different flaviviruses, including a recent West Nile virus isolate. Experiments were performed to define the stage in the viral lifecycle at which MPA abrogates infection. Early steps in viral infection, such as viral entry and nucleocapsid uncoating, were not the primary targets of MPA action since its inhibitory effect was retained when naked DV RNA was transfected directly into cells. Biosynthetic labeling experiments showed that MPA did not block the initial phase of viral translation but did interfere with viral protein synthesis in the amplification phase. Quantitative RT-PCR demonstrated that MPA prevented the accumulation of viral positive- and negative-strand RNA as the infection proceeded. We conclude that MPA inhibits flavivirus infection by preventing synthesis and accumulation of viral RNA.

Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium.

Human adenovirus type 12 (Ad12) infects human cells productively and leads to viral replication, whereas infection of hamster cells remains abortive, with total blocks in viral DNA replication and late viral gene transcription. The intranuclear fate of Ad12 DNA in productively infected human cells and in abortively infected hamster cells was monitored by using the fluorescent in situ hybridization (FISH) technique. Human HeLa cells, primary human umbilical cord fibroblasts, hamster BHK21 cells, primary embryonal hamster cells, and the Ad12-transformed T637 hamster cell line were studied. As early as 2 h after infection, extensive association of Ad12 DNA with metaphase chromosomes was demonstrated by FISH in all of these cells. Chromosomal association continued until late (24 to 28 h) after infection, when about 100% of the human cell nuclei and 70 to 80% of the hamster cell nuclei showed distinct FISH signals. This chromosomal association of Ad12 DNA in infected cells seemed to be rather firm, since it proved to be resistant to mechanically stretching the chromosomes and to different types of chemical treatment. Moreover, laser scan microscopy of mechanically stretched chromosomes from Ad12-infected HeLa cells and from the Ad12-transformed T637 cell line, with about 20 copies of Ad12 DNA provably integrated, revealed identical FISH patterns. Therefore, it was likely that even in infected cells the chromosomal association of Ad12 DNA was very similar to the integrated state. Late in productively infected cells, large nuclear areas were taken over by viral DNA replication, as visualized by FISH in interphase nuclei. Chromosomal association at many sites was frequently limited to one chromatid, but signals in adjacent positions on both chromatids were also seen. Upon the long-term cultivation and passage of abortively infected BHK21 cells for 96 h after infection, a gradual decrease of viral DNA association with chromosomes was observed. Integration of Ad12 DNA in hamster cells early after infection was previously documented, and recombination between viral and cellular DNAs in human cells was also shown. The FISH data on extensive chromosomal association of Ad12 DNA suggest a means to study the pathway of Ad12 DNA from early steps in viral infection via chromosomal interactions to integration events. In a different approach, Ad12 DNA, Ad12 DNA with the terminal protein covalently linked to its ends (Ad12 DNA-TP), or Ad2 DNA was simply added to the culture medium of HeLa or BHK21 cells. Precipitation or selection procedures were avoided. Depending on the experimental conditions, up to 25 to 30% of the interphase nuclei of HeLa cells and 9 to 19% of the interphase nuclei of BHK21 cells showed positive FISH signals at 24 h after the addition of DNA. Viral DNA also became associated in some cases with both chromatids. The uptake of Ad12 DNA-TP appeared to be 10 to 20 times more efficient than that of Ad12 DNA completely freed of proteins. Control bacteriophage lambda, M13, or plasmid DNA could not be detected in the nuclei under these conditions.