Inhibition Of Virus Spread Research Articles

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections.

Severe acute respiratory syndrome coronavirus‐2 (SARS CoV‐2) is the causative agent of the coronavirus disease‐2019 (COVID‐19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV‐2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID‐19. These results suggest that SARS CoV‐2 is able to spread from cell‐to‐cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA‐approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S‐n‐ and S‐o‐mediated cell fusion with complete inhibition at a 10‐μM concentration. In‐silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S‐n‐ and S‐o‐mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV‐2 symptoms appear.

ZAP's stress granule localization is correlated with its antiviral activity and induced by virus replication.

Cellular antiviral programs encode molecules capable of targeting multiple steps in the virus lifecycle. Zinc-finger antiviral protein (ZAP) is a central and general regulator of antiviral activity that targets pathogen mRNA stability and translation. ZAP is diffusely cytoplasmic, but upon infection ZAP is targeted to particular cytoplasmic structures, termed stress granules (SGs). However, it remains unclear if ZAP’s antiviral activity correlates with SG localization, and what molecular cues are required to induce this localization event. Here, we use Sindbis virus (SINV) as a model infection and find that ZAP’s localization to SGs can be transient. Sometimes no apparent viral infection follows ZAP SG localization but ZAP SG localization always precedes accumulation of SINV non-structural protein, suggesting virus replication processes trigger SG formation and ZAP recruitment. Data from single-molecule RNA FISH corroborates this finding as the majority of cells with ZAP localization in SGs contain low levels of viral RNA. Furthermore, ZAP recruitment to SGs occurred in ZAP-expressing cells when co-cultured with cells replicating full-length SINV, but not when co-cultured with cells replicating a SINV replicon. ZAP recruitment to SGs is functionally important as a panel of alanine ZAP mutants indicate that the anti-SINV activity is correlated with ZAP’s ability to localize to SGs. As ZAP is a central component of the cellular antiviral programs, these data provide further evidence that SGs are an important cytoplasmic antiviral hub. These findings provide insight into how antiviral components are regulated upon virus infection to inhibit virus spread.

Myeloid C-Type Lectin Receptors in Viral Recognition and Antiviral Immunity.

Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into host cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.

The brain parenchyma has a type I interferon response that can limit virus spread.

The brain has a tightly regulated environment that protects neurons and limits inflammation, designated "immune privilege." However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate-putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection.

A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells.

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel "dumbbell-shape" structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4(+)T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4(+)T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.

Oseltamivir inhibits influenza virus replication and transmission following ocular-only aerosol inoculation of ferrets

Ocular exposure to influenza virus represents an alternate route of virus entry capable of establishing a respiratory infection in mammals, but the effectiveness of currently available antiviral treatments to limit virus replication within ocular tissue or inhibit virus spread from ocular sites to the respiratory tract is poorly understood. Using an inoculation method that delivers an aerosol inoculum exclusively to the ocular surface, we demonstrate that oral oseltamivir administration following ocular-only aerosol inoculation with multiple avian and human influenza viruses protected ferrets from a fatal and systemic infection, reduced clinical signs and symptoms of illness, and decreased virus transmissibility to susceptible contacts when a respiratory infection was initiated. The presence of oseltamivir further inhibited influenza virus replication in primary human corneal epithelial cells. These findings provide critical experimental evidence supporting the use of neuraminidase inhibitors during outbreaks of influenza virus resulting in ocular disease or following ocular exposure.

Abstract 2639: Development of immunocytotoxic RNA therapeutics for human melanoma

Abstract Pattern recognition receptors (PRRs) are a pivotal component of anti-infectious immunity, and selective induction of PRR-mediated apoptosis in cancer cells represents a novel therapeutic strategy. Retinoic acid inducible gene-I (RIG-I) is a cytoplasmic PRR that recognizes virus-associated molecular patterns, such as uncapped 5′triphosphate (5′ppp) RNA. RIG-I activation triggers innate immune response and apoptosis in infected cells, resulting in the inhibition of viral spread. Interestingly, certain cancer cells are highly sensitive to RIG-I-mediated apoptosis, and treatment with RIG-I-stimulating RNAs can selectively kill cancer cells and facilitate the induction of anti-cancer immunity. Therefore, RIG-I-stimulating RNAs may prove valuable as immunostimulatory and anti-cancer agents. Because RNAs are extremely sensitive to serum nucleases, modified RNAs have been employed to increase the stability of RNA-based therapeutics. Such modifications, however, often abrogate the immunostimulatory activity of RNAs, which hinders their development as immunostimulatory agents. Herein, we demonstrate that short single-stranded RNAs (ssRNAs) (23-93 nucleotides) with 5′ppp and stem-loop structure(s) are a potent stimulus for type I interferon (IFN) production and apoptosis of human melanoma and prostate cancer cells. 2′fluoro (2′F) modification of these 5′ppp ssRNAs not only improves serum stability, but also enhances the ability of the RNAs to induce IFNβ production and apoptosis of human cancer cells, compared to their unmodified counterparts, while 2′ O-methyl modification abrogates the anti-cancer and IFN-inducing activities of 5′ppp ssRNAs. These 5′ppp 2′F ssRNAs elicit RIG-I- and mitochondrial antiviral-signaling protein-mediated IFN production and cytotoxic effects. This study suggests that such modified RNAs may represent a novel and effective anti-cancer therapeutic. Citation Format: Jaewoo Lee, Youngju Lee, Bruce A. Sullenger. Development of immunocytotoxic RNA therapeutics for human melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2014-2639

Tetherin can restrict cell-free and cell-cell transmission of HIV from primary macrophages to T cells.

Bst-2/Tetherin inhibits the release of HIV by tethering newly formed virus particles to the plasma membrane of infected cells. Although the mechanisms of Tetherin-mediated restriction are increasingly well understood, the biological relevance of this restriction in the natural target cells of HIV is unclear. Moreover, whether Tetherin exerts any restriction on the direct cell-cell spread of HIV across intercellular contacts remains controversial. Here we analyse the restriction endogenous Tetherin imposes on HIV transmission from primary human macrophages, one of the main targets of HIV in vivo. We find that the mRNA and protein levels of Tetherin in macrophages are comparable to those in T cells from the same donors, and are highly upregulated by type I interferons. Improved immunocytochemistry protocols enable us to demonstrate that Tetherin localises to the cell surface, the trans-Golgi network, and the macrophage HIV assembly compartments. Tetherin retains budded virions in the assembly compartments, thereby impeding the release and cell-free spread of HIV, but it is not required for the maintenance of these compartments per se. Notably, using a novel assay to quantify cell-cell spread, we show that Tetherin promotes the transfer of virus clusters from macrophages to T cells and thereby restricts the direct transmission of a dual-tropic HIV-1. Kinetic analyses provide support for the notion that this direct macrophage-T cell spread is mediated, at least in part, by so-called virological synapses. Finally, we demonstrate that the viral Vpu protein efficiently downregulates the cell surface and overall levels of Tetherin, and thereby abrogates this HIV restriction in macrophages. Together, our study shows that Tetherin, one of the most potent HIV restriction factors identified to date, can inhibit virus spread from primary macrophages, regardless of the mode of transmission.

Retinoic Acid-inducible Gene-I and Interferon-β Promoter Stimulator-1 Augment Proapoptotic Responses Following Mammalian Reovirus Infection via Interferon Regulatory Factor-3

During viral infection, cells initiate antiviral responses to contain replication and inhibit virus spread. One protective mechanism involves activation of transcription factors interferon regulatory factor-3 (IRF-3) and NF-kappaB, resulting in secretion of the antiviral cytokine, interferon-beta. Another is induction of apoptosis, killing the host cell before virus disseminates. Mammalian reovirus induces both interferon-beta and apoptosis, raising the possibility that both pathways are initiated by a common cellular sensor. We show here that reovirus activates IRF-3 with kinetics that parallel the activation of NF-kappaB, a known mediator of reovirus-induced apoptosis. Activation of IRF-3 requires functional retinoic acid inducible gene-I and interferon-beta promoter stimulator-1, but these intracellular sensors are dispensable for activation of NF-kappaB. Interferon-beta promoter stimulator-1 and IRF-3 are required for efficient apoptosis following reovirus infection, suggesting a common mechanism of antiviral cytokine induction and activation of the cell death response.

Rapid production of neutralizing antibody leads to transient hepadnavirus infection.

Hepatitis B virus (HBV) frequently causes transient infections in adults but chronic infections in infants. The basis of these age-related outcomes is not known. Infection of ducks with duck hepatitis B virus (DHBV) displays a similar dependence of outcome on the age of the host at the time of infection. In this study we compared the infection of ducks at 3 days and 3 weeks of age. We found that the efficiency of infection of hepatocytes by virus in the inoculum was similar between the two age groups but that spread of the infection throughout the liver was severely inhibited in the 3-week-old-old ducks, while a rapid spread of the infection was observed in 3-day-old ducklings. Inhibition of virus spread was accompanied by the appearance in the serum of virus neutralizing activity, as assayed by blocking of infection of primary hepatocyte cultures. Neutralizing activity appeared as early as 1 or 2 days postinfection and increased during the next 2 weeks. Depletion of immunoglobulins from serum eliminated the neutralizing activity. The specific depletion of IgM indicated that IgM appeared as the dominant fraction of neutralizing antibody in the first 2 days postinfection, but declined from day 3 on while IgG antibody rose. We conclude that excess neutralizing antibody arising rapidly in birds inoculated at 3 weeks of age but not in newly hatched ducks prevented secondary cycles of infection, resulting in a limited infection in the liver and contributing to the eventual transient outcome of the infection.

Human mannan-binding lectin inhibits the infection of influenza A virus without complement.

Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen-like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Monoclonal antibodies to p24-core protein of HIV-1 mediate ADCC and inhibit virus spread in vitro

Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies. To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells. Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro. The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures. Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Inhibition by iota-carrageenan of the spread of murine cytomegalovirus from the peritoneal cavity to the blood plasma

The mechanism of iota-carrageenan (i-CAR)-induced protection against murine cytomegalovirus (MCMV) infection of mice was analysed. The virus titres in the target organs on the fourth day correlated with that in the plasma at 1 h after intraperitoneal inoculation of MCMV, irrespective of i-CAR treatment. Pretreatment of mice with i-CAR induced infiltration of polymorphonuclear neutrophils into the peritoneal cavity and inhibition of viral spread from the peritoneal cavity to the plasma. Although the direct relationship of these two phenomena was unclear, the inhibition by i-CAR of viral spread resulted in protection against MCMV infection of mice.

Protective anti-reovirus monoclonal antibodies and their effects on viral pathogenesis

We used a recently isolated and characterized panel of monoclonal antibodies (MAbs) specific for cross-reactive determinants on reovirus outer capsid proteins to define mechanisms of antibody-mediated protection in vivo. We studied the capacities of MAbs to protect against lethal infection with reoviruses which differ in site of primary replication, route of spread, and central nervous system tropism. We found the following. (i) MAbs specific for each of the viral outer capsid proteins (sigma 1, sigma 3, and mu 1) and the core spike protein (lambda 2) were protective under certain circumstances. (ii) In vitro properties of MAbs, including isotype, neutralization of viral infectivity, inhibition of virus-induced hemagglutination, and avidity of binding, were poorly predictive of the capacities of MAbs to protect in vivo. (iii) MAbs did not act at a single stage during pathogenesis to mediate protection; instead, protective MAbs were capable of altering a variety of stages in reovirus pathogenesis. (iv) MAbs protective against one reovirus also protected against other reoviruses that utilized different pathogenetic strategies, suggesting that the viral epitope bound by an antibody rather than the pathogenetic strategy employed by the virus is a critical determinant of antibody-mediated protection in vivo. (v) A prominent mechanism of protective MAb action is inhibition of viral spread through nerves from a site of primary replication (e.g., the intestine or muscle tissue) to the central nervous system.

Delineation of putative mechanisms involved in antibody-mediated clearance of rabies virus from the central nervous system.

The in vitro biological activities of several rabies virus-neutralizing monoclonal antibodies (mAbs) were compared with their ability to prevent a lethal rabies virus encephalomyelitis. The protective activity of a particular mAb in vivo did not correlate with its virus-neutralizing activity in vitro; rather it was related to the mAb's ability to inhibit virus spread from cell to cell and to restrict rabies virus RNA transcription. Since treatment of rabies virus-infected cells with virus-neutralizing mAbs results in an endocytosis of the antibody, we hypothesize that an antibody may exert its inhibitory activity even after uptake by the cell. Post-exposure treatment of rats with a mAb that inhibited both virus spread and virus RNA transcription in vitro resulted in viral clearance from the central nervous system and protected the animals against a lethal rabies virus infection.

Protease Inhibitors as Potential Therapeutic Agents for AIDS

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus

We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread. We also detected variations in the concentrations of drug needed to inhibit virus replication in different cell types. Higher concentrations of drug were needed to inhibit virus replication in chicken embryo fibroblasts than in the continuous quail cell line QT6. Viral DNA synthesis in infected cells was shown to be inhibited in the presence of the drug. The triphosphate form of the analog acted as a competitive inhibitor of purified viral reverse transcriptase, with a Ki of 0.09 +/- 0.003 microM, and was incorporated as a chain terminator during reverse transcription of the natural viral RNA substrate in vitro.

Effect of 3′-azido-2′,3′-dideoxythymidine (AZT) and neutralizing antibody on human immunodeficiency virus (HIV)-induced cytopathic effects: Implication of giant cell formation for the spread of virus in vivo

The effect of 3′-azido-2′,3′-dideoxythymidine (AZT) on the human immunodeficiency virus (HIV)-associated giant cell formation was studied in vitro. For this purpose we developed a coculture system using Molt-4 and its virus-producing cell, Molt-4/HTLV-III, which induced syncytia very efficiently. Treatment of the cocultures with 1 and 5 μ M of AZT did not inhibit induction of multinucleated giant cells, although only 0.1 μ M AZT resulted in almost complete inhibition of HIV replication in Molt-4 cells by cell-free virus infection. This was also evidenced by the assays for viral antigen-positive cells, reverse transcriptase activity, and virus particles released from cell cultures after AZT treatment. When the cocultures were treated with 1% neutralizing antibody (NA) from HIV-infected individuals alone, giant cell formation was inhibited to some extent. However, the concomitant treatment of culture with AZT and NA resulted in much stronger inhibition of giant cell formation. The amount of CD4 antigens on the surface of cells was reduced greatly in the HIV producer cells (Molt-4, H9, and MT-4 cells) as compared to their HIV-free counterparts. These data suggest that (1) both CD4 antigen and viral antigens on the surface of cells play central roles in the induction of multinucleated giant cells and (2) AZT is more effective in inhibition of viral spread in patients with higher NA, probably at an earlier stage of the disease than in patients with lower NA titer.

Ultrastructural Studies on Potato Phloem Cells Infected with Potato Leaf Roll Virus—Comparison of Two Potato Varieties

Abstract The sequence of cytopathological changes in the phloem of two potato cultivars, Osa and Apta, after infection by potato leaf roll virus (PLRV), was investigated. Osa is susceptible but tolerant to the virus, whereas Apta is resistant but sensitive. The cytopathological differences between the two potato varieties concern mainly the range of the apoplast changes in the parts of the phloem that are involved in the systemic spread of the infection in the plant. The sieve tubes of Apta, into which the virus particles are translocated, respond by intensive formation of callose, which results in their necrosis and inhibition of virus spread. In the case of Osa the callose reaction of the sieve tube was less intensive. It is assumed, on the basis of the present investigation, that the ability of the potato plant to establish apoplastic defence against the spread of the virus particles varies depending on the genotype of the cultivar.