Increased Virus Production Research Articles

Regulation of human immunodeficiency virus replication by 2',5'-oligoadenylate-dependent RNase L.

Activation of RNase L by 2',5'-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.

HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo.

The human immunodeficiency virus type 1 (HIV-1) encodes a protein, called Vpr, that prevents proliferation of infected cells by arresting them in G2 of the cell cycle. This Vpr-mediated cell-cycle arrest is also conserved among highly divergent simian immunodeficiency viruses, suggesting an important role in the virus life cycle. However, it has been unclear how this could be a selective advantage for the virus. Here we provide evidence that expression of the viral genome is optimal in the G2 phase of the cell cycle, and that Vpr increases virus production by delaying cells at the point of the cell cycle where the long terminal repeat (LTR) is most active. Although Vpr is selected against when virus is adapted to tissue culture, we show that selection for Vpr function in vivo occurs in both humans and chimpanzees infected with HIV-1. These results suggest a novel mechanism for maximizing virus production in the face of rapid killing of infected target cells.

Increased Virus Production in Suspension Culture by a Trichoplusia ni Cell Line in Serum-Free Media

Trichoplusia ni-derived BTI-Tn5B1–4 (Tn-5B1–4) cells were grown in 50 mL spinner flasks in four different formulations of serum-free medium (SFM) . The objectives of this work were to determine the capacity of this cell line to grow and support virus replication in suspension culture in different SFM. Maximum cell densities exceeding 3.0 × 106 cells/mL were attained for all cell/medium combinations. Likewise, cell doubling times were all within 20–22 h. Infection studies with a wild-type isolate of Autographa californicanuclear polyhedrosis virus (AcMNPV) yielded between 156 and 187 occlusion bodies (OBs) /cell. The lethal effect of AcMNPV-OBs produced in this cell line in the various types of media was demonstrated in vivousing T. ni neonate larvae. The percent mortality at a single dose (1.0 × 106 OBs/mL) ranged from 40 to 60% at 4 days post-inoculation (p.i.) . These results compared favorably with mortality rates found for AcMNPV-OBs produced in vivo.DNA quantification analysis, comparing polyhedra-derived viral DNA concentrations from an equal number of cell culture- and larval-derived OBs, was also determined. The larval-derived OB DNA concentration averaged 0.514 μg/108 OBs, whereas the cell culture-derived viral DNA concentrations ranged from 0.347 to 0.710 μg/108 OBs. These results suggest that no significant differences exist in DNA content between larvae- and cell culture-derived samples. Expression of the recombinant protein, secreted alkaline phosphatase (SEAP), ranged from 2.7 to 5.5 IU/mL for each cell/medium combination, which was consistent with previous static culture results for this same cell line in serum-containing TNM-FH medium. Electron microscopic analysis of OBs produced in vivo or in vitro revealed no obvious differences in packaging or number of enveloped nucleocapsids per OB between samples. However, AcMNPV-OBs from infected larvae appeared physically smaller (1.28 ± 0.164 μm) than those produced in cell culture (1.72 ± 0.120) . The Tn-5B1–4 cell line is adaptable to suspension culture in serum-free medium and is suitable for further development in the production of viruses and recombinant proteins on an industrial scale.

Kinetics of viral RNA synthesis following cell-to-cell transmission of human immunodeficiency virus type 1.

The temporal appearance and levels of human immunodeficiency virus type 1 (HIV-1) tat, rev, nef, env and gag mRNA species were examined using a synchronized, one-step, cell-to-cell HIV-1 infection model involving HUT-78 cells and HIV- 1 persistently infected H3B cells. Individual mRNAs were quantified by RT-PCR using RNA standards transcribed in vitro from cDNA clones. Consistent with an infection that produces high yields of virus, significant levels of env and gag mRNAs were detected in the cytoplasm of infected cells late in the infection cycle. However, at no time after infection did levels of tat, rev and nef mRNA, which encode the regulatory proteins of HIV-1, exceed their levels present in the persistently infected virus donor H3B cells. The absence of early phase induction of these mRNAs is in contrast to what is observed in cell-free HIV-1 infections or in PMA-stimulated HIV-1 chronically infected cell lines. Our results suggest that tat and rev mRNAs are already present in the cytoplasm of the persistently infected virus donor cells at levels sufficient for initiation and establishment of a highly productive infection in HIV-1 fusion-mediated infected cells. Thus, lack of sufficient Tat and Rev proteins is not likely to be the limiting factor for virus production in H3B cells, nor is increased production of these proteins likely to be the cause of the increased virus production seen following cell-to-cell transmission.

Entry of Mouse Hepatitis Virus into Cells by Endosomal and Nonendosomal Pathways

OBLV60 is an acid-dependent syncytium-forming variant isolated from OBL21 cells persistently infected with the pH-independent mouse hepatitis virus (MHV)-4 strain. The fusion activity of OBLV60 can be strictly regulated by controlling pH and thus provides the means to definitively examine the entry of MHV into cells by endosomal and nonendosomal pathways. Shortly after high multiplicity infection, both MHV-4 and OBLV60 were detected by electron microscopy in endosomal vesicles and were recovered from lysates of cells treated with proteinase K to remove extracellular virus. For OBLV60, but not MHV-4, exposure to lysosomotropic compounds early in infection prevented viral penetration and significantly reduced viral yields. These results suggested that both MHV-4 and OBLV60 utilized the endosomal route of entry into cells, but that MHV-4 did not require acidification of endosomal vesicles. Studies on the entry of virus through fusion at the cell surface were performed by briefly exposing surface-bound OBLV60 to a fusion-permissive pH under conditions that prevent endocytic entry. Acid treatment of surface-bound OBLV60 caused a significant increase in the yields of virus produced in cultures of fusion-sensitive Sac− or DBT cells, demonstrating entry of virus by fusion at the cell surface. No measurable increase in virus production was detected with acid treatment of OBLV60 bound to OBL21 cells, suggesting that entry at the cell surface does not occur in these cells, which are resistant to MHV-induced syncytia formation. These results raise interesting questions concerning how mechanisms of MHV entry influence the selection of fusion variants.

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins.

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation.

After integration in the host cell genome, the HIV-1 provirus is packaged into chromatin. A specific chromatin disruption occurs in the HIV-1 promoter during transcriptional activation in response to TNF-alpha, suggesting that chromatin plays a repressive role in HIV-1 transcription and that chromatin modification(s) might result in transcriptional activation. We have treated several cell lines latently infected with HIV-1 with two new specific inhibitors of histone deacetylase, trapoxin (TPX) and trichostatin A (TSA), to cause a global hyperacetylation of cellular histones. Treatment with both drugs results in the transcriptional activation of the HIV-1 promoter and in a marked increase in virus production. Dose-response curves and kinetic analysis show a close correlation between the level of histone acetylation and HIV-1 gene expression. In contrast, both TPX and TSA have little or no effect on HIV-1 promoter activity following transient transfection of an HIV-1 promoter-reporter plasmid. Activation of HIV-1 transcription by TSA and TPX treatment occurs in the absence of NF-kappa B induction. Chromatin analysis of the HIV-1 genome shows that a single nucleosome (nuc-1) located at the transcription start and known to be disrupted following TNF-alpha treatment, is also disrupted following TPX or TSA treatment. This disruption is independent of transcription as it is resistant to alpha-amanitin. These observations further support the crucial role played by nuc-1 in the suppression of HIV-1 transcription during latency and demonstrate that transcriptional activation of HIV-1 can proceed through a chromatin modification.

Use of sodium butyrate to enhance production of retroviral vectors expressing CFTR cDNA.

Previously, we constructed a retrovirus vector (LCFSN) for transduction and expression of the cDNA encoding the normal human cystic fibrosis transmembrane conductance regulator (CFTR). The titer of virus from amphotropic packaging cells producing the LCFSN vector was low (10(3)-10(4) infectious units/ml). In an attempt to increase virus production, we used sodium butyrate (NaB) to treat murine retrovirus packaging cells producing this vector. NaB treatment increased the production of LCFSN from between 20-fold to greater than 1,000-fold, depending upon the producer clone, thereby resulting in virus titers up to about 1 x 10(7) infectious units/ml. This induction of virus titer could be accounted for, at least in part, by an increase in steady-state levels of full-length vector RNA within the producer cells. With some clonal producer cell lines, lowering the temperature of the virus harvest in combination with NaB treatment resulted in an apparent synergistic increase in virus production. The production of retrovirus vectors containing genes other than CFTR could also be increased by NaB treatment, although the enhancement in titer was modest (2-fold to 10-fold). The increase in virus production was not accompanied by an induction of replication-competent helper virus. NaB treatment also increased the transient production of retroviral vectors following DNA-mediated transfection into packaging cells such that virus titers of greater than 10(6) infectious units/ml could be readily attained.

Regulation of human immunodeficiency virus type 1 and cytokine gene expression in myeloid cells by NF-kappa B/Rel transcription factors

CD4+ macrophages in tissues such as lung, skin, and lymph nodes, promyelocytic cells in bone marrow, and peripheral blood monocytes serve as important targets and reservoirs for human immunodeficiency virus type 1 (HIV-1) replication. HIV-1-infected myeloid cells are often diminished in their ability to participate in chemotaxis, phagocytosis, and intracellular killing. HIV-1 infection of myeloid cells can lead to the expression of surface receptors associated with cellular activation and/or differentiation that increase the responsiveness of these cells to cytokines secreted by neighboring cells as well as to bacteria or other pathogens. Enhancement of HIV-1 replication is related in part to increased DNA-binding activity of cellular transcription factors such as NF-kappa B. NF-kappa B binds to the HIV-1 enhancer region of the long terminal repeat and contributes to the inducibility of HIV-1 gene expression in response to multiple activating agents. Phosphorylation and degradation of the cytoplasmic inhibitor I kappa B alpha are crucial regulatory events in the activation of NF-kappa B DNA-binding activity. Both N- and C-terminal residues of I kappa B alpha are required for inducer-mediated degradation. Chronic HIV-1 infection of myeloid cells leads to constitutive NF-kappa B DNA-binding activity and provides an intranuclear environment capable of perpetuating HIV-1 replication. Increased intracellular stores of latent NF-kappa B may also result in rapid inducibility of NF-kappa B-dependent cytokine gene expression. In response to secondary pathogenic infections or antigenic challenge, cytokine gene expression is rapidly induced, enhanced, and sustained over prolonged periods in HIV-1-infected myeloid cells compared with uninfected cells. Elevated levels of several inflammatory cytokines have been detected in the sera of HIV-1-infected individuals. Secretion of myeloid cell-derived cytokines may both increase virus production and contribute to AIDS-associated disorders.

Anti-AIDS (acquired immune deficiency syndrome) agents. 17. New brominated hexahydroxybiphenyl derivatives as potent anti-HIV agents.

Sixteen biphenyl derivatives were synthesized and evaluated for their inhibitory activity against HIV-1 replication in acutely infected H9 cells. 3-Bromo- (4) and 3,3'-dibromo-4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2,2'- bis(methoxycarbonyl)biphenyl (5) demonstrated potent anti-HIV activity with EC50 values of 0.52 and 0.23 micrograms/mL and therapeutic index values of > 190 and > 480, respectively. A comparison of the anti-HIV activity of these biphenyl derivatives suggested that the types of substituents on the phenolic hydroxy groups rather than the number of bromine(s) on the aromatic rings are important to the enhanced anti-HIV activity. Compounds 4 and 5 also showed potent inhibitory activity against HIV-1 reverse transcriptase in a template-primer dependent manner. The site of inhibition of HIV could be related to inhibition of this enzyme. Compounds 4 and 5 did not induce virus expression from the chronic HIV-1-infected cell lines ACH-2 and U1. Furthermore, these two agents did not inhibit an increase in virus production from the chronic HIV-1-infected cell lines when the phorbol ester PMA was present.

Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic.

Regulation of human immunodeficiency virus type 1 and cytokine gene expression in myeloid cells by NF-kappa B/Rel transcription factors.

CD4+ macrophages in tissues such as lung, skin, and lymph nodes, promyelocytic cells in bone marrow, and peripheral blood monocytes serve as important targets and reservoirs for human immunodeficiency virus type 1 (HIV-1) replication. HIV-1-infected myeloid cells are often diminished in their ability to participate in chemotaxis, phagocytosis, and intracellular killing. HIV-1 infection of myeloid cells can lead to the expression of surface receptors associated with cellular activation and/or differentiation that increase the responsiveness of these cells to cytokines secreted by neighboring cells as well as to bacteria or other pathogens. Enhancement of HIV-1 replication is related in part to increased DNA-binding activity of cellular transcription factors such as NF-kappa B. NF-kappa B binds to the HIV-1 enhancer region of the long terminal repeat and contributes to the inducibility of HIV-1 gene expression in response to multiple activating agents. Phosphorylation and degradation of the cytoplasmic inhibitor I kappa B alpha are crucial regulatory events in the activation of NF-kappa B DNA-binding activity. Both N- and C-terminal residues of I kappa B alpha are required for inducer-mediated degradation. Chronic HIV-1 infection of myeloid cells leads to constitutive NF-kappa B DNA-binding activity and provides an intranuclear environment capable of perpetuating HIV-1 replication. Increased intracellular stores of latent NF-kappa B may also result in rapid inducibility of NF-kappa B-dependent cytokine gene expression. In response to secondary pathogenic infections or antigenic challenge, cytokine gene expression is rapidly induced, enhanced, and sustained over prolonged periods in HIV-1-infected myeloid cells compared with uninfected cells. Elevated levels of several inflammatory cytokines have been detected in the sera of HIV-1-infected individuals. Secretion of myeloid cell-derived cytokines may both increase virus production and contribute to AIDS-associated disorders.

Lectin-mediated enhancement of dengue virus infection in a mouse macrophage cell line Mk1.

Treatment of a mouse macrophage cell line Mk1 with pokeweed mitogen (PWM) either before or during but not after virus inoculation resulted in an enhancement of dengue virus (DV) infection. The infection enhancement was primarily due to an increase in the number of DV-infected cells but not to increased virus production in a cell. These results suggested that PWM treatment mediated increased DV binding and/or penetration to Mk1 cells, thereby resulting in the infection enhancement. N-acetylglucosamine (GlucNAc) did not suppress PWM-mediated enhancement of DV infection when added to Mk1 cells after PWM treatment was done, although GlucNAc clearly suppressed the effect of PWM when added simultaneously with PWM. The results implied the possibility that the PWM-mediated increase in viral binding/penetration was not due to a cross-linking by PWM between DV and a cell-surface receptor, but due to another mechanism, presumably exposure of a masked DV receptor(s). The DV receptor, unidentified as yet, involved in the PWM-mediated infection enhancement appeared to have no relation with IgG Fc receptors that are known to be involved in antibody-mediated enhancement of DV infection.

Single Amino Acid Change in Tat Determines the Different Rates of Replication of Two Sequential HIV-1 Isolates

Human immunodeficiency virus type 1 (HIV-1) isolates display differences in their patterns of replication in vitro. Previous studies with recombinant viruses generated between two sequential HIV-1 isolates that differ in replication rates have shown that the determinant for this biological property maps to a region of the viral genome that encompasses the tat gene. In the present study, we examined if there is a functional difference in the Tat protein of the two isolates. We observed that the level of transactivation induced by Tat of the fast replicating, highly cytopathic virus (HIV-1 SF13mc) was three- to eightfold higher than the level seen by the Tat of the slow replicating virus (HIV-1 SF2mc). Furthermore, a single amino acid mutation in the HIV-1 SF13mc Tat leads to reduced transactivation activity of the mutant protein and a delayed replication rate of the mutant virus. Thus, selection of a Tat variant with higher transactivation potential could be responsible for the increased virus production that is often associated with a pathogenic HIV strain.

Glucocorticoids synergize with tumor necrosis factor alpha in the induction of HIV expression from a chronically infected promonocytic cell line.

In this study we have investigated the effects of glucocorticoids (GCs) on the expression of human immunodeficiency virus (HIV) in a chronically infected promonocytic cell line, U1. Although no increase in virus production was observed in U1 cells stimulated with physiological concentrations of GC alone, costimulation with dexamethasone plus tumor necrosis factor alpha (TNF-alpha) synergistically enhanced TNF-alpha-dependent HIV expression. Molecular analysis demonstrated that GCs plus TNF-alpha resulted in an accumulation of steady state HIV RNA secondary to either an increase in transcription or an increase in message stability. These findings may be of physiological relevance because GCs are used in the treatment of certain disorders associated with HIV infection and TNF-alpha levels have been reported to be elevated in the plasma and cerebrospinal fluid of certain HIV-infected individuals.

Soluble tumor necrosis factor receptor: inhibition of human immunodeficiency virus activation.

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to stimulate human immunodeficiency virus type 1 (HIV-1) replication in both chronically and acutely infected T lymphocytes and monocytes. Transcriptional activation of the HIV long terminal repeat and subsequent increase in virus production are linked to TNF activation of the cellular transcription factor NF-kappa B. Here we report the use of two forms of soluble recombinant type 1 (p80) TNF receptor to inhibit TNF-induced HIV activation in vitro. One receptor form is a monomer containing the entire 236 residues of the extracellular (ligand-binding) portion of p80. A second receptor form is a chimeric homodimer containing these residues fused to a truncated human IgG1 immunoglobulin heavy chain and, thus, resembles a bivalent antibody without light chains. These recombinant receptor proteins were tested for their ability to inhibit TNF-alpha-induced expression of HIV-1 in chronically infected human cell lines. We also examined the ability of the soluble receptors to limit the activation of the HIV-long terminal repeat transcription. The soluble TNF receptor dimer was most effective at blocking TNF-alpha-induced HIV-1 expression in both monocytoid and lymphoid cells. The molar ratio of TNF-receptor dimer to TNF-alpha found to be most effective was, at least, 5:1. We conclude that at specific TNF/soluble TNF-receptor dimer ratios, TNF-alpha-induced HIV-1 transcription and expression can be limited in vitro.

Human Immunodeficiency Virus-1 Infection of the Human Promyelocytic Cell Line HL-60: High Frequency of Low-Level Infection and Effect of Subsequent Cell Differentiation

AbstractAs a model system to study the infection of early myeloid cells by human immunodeficiency virus-1 (HIV-1), we have infected the human promyelocytic cell line, HL-60, with a recombinant selectable HIV-1 clone. A fully infected population showed a relatively high frequency of low-level infection, with 40% of subcloned cells being negative by reverse transcriptase and p24 indirect immunofluorescence analysis and displaying only low levels of supernatant p24. The same treatment of a T-lymphoid cell line produced 100% productive infections. HIV-1 infection of HL-60 did not appear to alter the state of differentiation of the cells, as assessed by surface antigen expression, regardless of the level of viral expression. Furthermore, infected cells were able to respond normally to chemical inducers of differentiation. Induction of differentiation towards monocyte/macrophages by phorbol myristate acetate activated the HIV-1 long terminal repeat in a transient transfection system, and there was a corresponding increase in viral production from the infected subclones. Granulocytic differentiation, as stimulated by dimethyl sulfoxide or retinoic acid, had no effect on long terminal repeat activity and did not stimulate viral replication. These data suggest that low-level HIV-1 infections may be established at a relatively high frequency in myeloid precursor cells, and that different pathways of promyelocytic differentiation vary in their ability to stimulate HIV-1 replication.

Human immunodeficiency virus-1 infection of the human promyelocytic cell line HL-60: high frequency of low-level infection and effect of subsequent cell differentiation

As a model system to study the infection of early myeloid cells by human immunodeficiency virus-1 (HIV-1), we have infected the human promyelocytic cell line, HL-60, with a recombinant selectable HIV-1 clone. A fully infected population showed a relatively high frequency of low-level infection, with 40% of subcloned cells being negative by reverse transcriptase and p24 indirect immunofluorescence analysis and displaying only low levels of supernatant p24. The same treatment of a T-lymphoid cell line produced 100% productive infections. HIV-1 infection of HL-60 did not appear to alter the state of differentiation of the cells, as assessed by surface antigen expression, regardless of the level of viral expression. Furthermore, infected cells were able to respond normally to chemical inducers of differentiation. Induction of differentiation towards monocyte/macrophages by phorbol myristate acetate activated the HIV-1 long terminal repeat in a transient transfection system, and there was a corresponding increase in viral production from the infected subclones. Granulocytic differentiation, as stimulated by dimethyl sulfoxide or retinoic acid, had no effect on long terminal repeat activity and did not stimulate viral replication. These data suggest that low-level HIV-1 infections may be established at a relatively high frequency in myeloid precursor cells, and that different pathways of promyelocytic differentiation vary in their ability to stimulate HIV-1 replication.

A novel method for increased yield of immunocompetent virus for vaccine production

Cultured human cells exposed to the pesticide emulsifier Atlox, 6 to 8 h prior to Infection with influenza A virus, increased virus production approximately 10-fold. Antibodies against the 'enhanced virus' neutralized plaque formation and reacted equally well with non-enhanced virus in serological tests (haemagglutination-inhibition and radioimmunoassays). The procedure has great potential in cutting costs of production for some virus vaccines.

Cytokine enhancement of simian immunodeficiency virus (SIV/mac) from a chronically infected cloned T-cell line (HuT-78)

Simian immunodeficiency viruses (SIV) are a family of primate lentiviruses similar to human immunodeficiency viruses (HIV) in their genetic sequence and pathogenesis. However, host-derived cofactors which may determine the extent of viral replication are not clearly defined for SIV or HIV infections. A HuT-78 cell line chronically infected with SIV/mac strain 251, was biologically cloned and characterized for the ability to produce infectious viral particles, viral structural protein profile, cellular antigen surface phenotype and tested to determine the effects of recombinant cytokines on SIV replication. Reverse transcriptase (RT) assay was used to measure the replication of SIV/mac in response to various concentrations of recombinant cytokines (1-1000 units/ml). We report that tumor necrosis factor-alpha (rTNF-alpha), gamma-interferon (rIFN-gamma), interleukin 2 (rIL-2), and granulocyte-macrophage colony stimulating factor (rGM-CSF) induced approximately a 2 to 3 fold increase in virus RT activity compared with untreated SIV-infected HuT-78 cells. In contrast, viral replication was not enhanced or minimally enhanced by interleukin 1 (rIL-1), interleukin 3 (rIL-3), or interleukin 4 (rIL-4) at similar dosages. Furthermore, SIV replication in response to rTNF-alpha and rIFN-gamma occurred in a dose dependent fashion. These data suggest that SIV-infected T-lymphocyte lines are responsive to particular cytokines resulting in increased virus production.