Induction of latent human cytomegalovirus by conventional gamma irradiation and prevention by treatment with INACTINE PEN110.

BACKGROUND AND OBJECTIVES: Two different leucocyte-inactivation technologies--gamma irradiation and INACTINE PEN110--were evaluated for their effects on cell-associated human cytomegalovirus (CMV). MATERIALS AND METHODS: In vitro CMV-infected cells were spiked into leucoreduced red blood cell concentrates (RCC) or medium at a final concentration of 0.5 - 1 x 10(7) cells/ml to mimic non-leucoreduced levels of leucocytes. The spiked RCC/medium was divided into three equal units and treated with gamma irradiation at the US Food and Drug Administration (FDA)-approved dose of 25 Gy, with 0.1% v/v PEN110 at 22 degrees C for 24 h, or stored at 4 degrees C as a control. The treated and control cells were recovered and tested using infectivity, viability and polymerase chain reaction (PCR) assays. RESULTS: Gamma-irradiated CMV-infected cells produced active virus, as shown by both infectivity assays and PCR quantification of viral DNA. PCR analysis demonstrated higher CMV DNA levels in gamma-irradiated, latently infected monocytic THP-1 cells than untreated control cells. The increased virus production in gamma-irradiated cells was paralleled by an increased metabolic rate and the development of enlarged multinuclear cells. In contrast, PEN110 treatment terminated virus replication and completely inactivated the infected cell. CONCLUSIONS: These results demonstrate that gamma irradiation, at levels currently used to treat RCC, has the capacity to induce expression of CMV, whereas PEN110 inhibits CMV replication and efficiently inactivates the infected cells.

Broad-spectrum virus reduction in red cell concentrates using INACTINE PEN110 chemistry.

BACKGROUND AND OBJECTIVES: The risk of transmission of blood-borne pathogens by transfusion is a persistent problem in medicine. To address this safety issue, INACTINE PEN110 chemistry is being utilized to develop a process for preparing pathogen-reduced red blood cell concentrates (RBCC). The purpose of this study was to characterize the virucidal effectiveness of the INACTINE PEN110 chemistry in full units of RBCC by using a panel of viruses with diverse properties in composition, size and shape. MATERIALS AND METHODS: The panel included four enveloped (bovine viral diarrhoea virus, pseudorabies virus, vesicular stomatitis Indiana virus and sindbis virus), six non-enveloped (porcine parvovirus, human adenovirus 2, reovirus 3, vesicular exanthema of swine virus, bluetongue virus, and foot and mouth disease virus) and cell-associated (human immunodeficiency) viruses. All viruses were individually spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC units and treated with 0.1% PEN110 (vol/vol) at 22 +/- 2 degrees C for up to 22 +/- 2 h. The PEN110 treatment reaction was stopped by chemical quenching, and residual virus was assayed. The cytotoxicity effect of PEN110-treated RBCC on indicator cells and the potential interference with the ability of the virus to infect indicator cells was determined and taken into consideration for calculating the virus-reduction factors, to avoid underestimation or overestimation of the virus reduction. RESULTS: The kinetics of inactivation for viruses spiked into CPD/AS-1, CP2D/AS-3 and CPD/AS-5 RBCC were equivalent. All viruses analysed in this study were reduced to the limit of detection of the assay. The reduction factors for the virus panel ranged from 4.2 to 7.5 log10/ml. CONCLUSIONS: The results from the study demonstrate for the first time that a pathogen-reduction technology for RBCC can achieve a broad-spectrum virucidal effect against both enveloped and non-enveloped viruses. The broad spectrum of virucidal activity of INACTINE PEN110, and equivalent kinetics of virus inactivation in RBCC prepared using different commercially available RBC storage solutions, demonstrate the robustness of this pathogen-reduction process.

Role of Vif in stability of the human immunodeficiency virus type 1 core.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription. vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.

Isolation of human immunodeficiency virus type 1 cores: retention of Vpr in the absence of p6(gag).

Mature human immunodeficiency virus type 1 (HIV-1) virions contain a typically cone-shaped core that encases the viral genome. In this study, we established conditions which allowed the efficient isolation of morphologically intact HIV-1 cores from virions. The isolated cores consisted mostly of cones which appeared uniformly capped at both ends but were heterogeneous with respect to the shape of the broad cap as well as the dimensions and angle of the cone. Vpr, a nonstructural virion component implicated in the nuclear import of the viral genome, was recovered in core preparations of HIV-1 and simian immunodeficiency viruses from African green monkeys. Unexpectedly, p6(gag), a structural protein required for the incorporation of Vpr, was absent from HIV-1 core preparations. Taken together, our results indicate that the incorporation of Vpr into the virion core is a conserved feature of primate lentiviruses and that the interactions required for the uptake of Vpr into assembling particles differ from those which confine Vpr within the core.

Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus.

Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells. The human cytomegalovirus US28 gene encodes a chemokine receptor homolog that has been reported to function as an HIV-1 coreceptor. However, studies of US28 have given conflicting results regarding its ability to mediate HIV-1 entry. We examined the ability of US28 to function as an HIV-1 coreceptor in various cell lines and found that its coreceptor activity is highly cell dependent. US28 could function as a coreceptor for HIV-1 entry in HeLa and U87 cells but not in COS-1 and Cf2Th cells. In COS-1 cells, US28 was expressed on the cell surface and could mediate cell-cell fusion with HIV-1 Env-expressing cells, suggesting that the block to infection may result from a defect in virus internalization or postentry steps. In Cf2Th cells, US28 was expressed at high levels intracellularly but was not transported to the cell surface. The block in US28 coreceptor function in COS-1 and Cf2Th cells was coreceptor dependent, since CCR5, CXCR4, and other coreceptors can mediate HIV-1 entry in these cell lines. HIV-1 viruses pseudotyped with the MuLV or VSV Env entered and replicated at similar efficiency in COS-1 and U87 cells in single-cycle infections, suggesting that postentry and other early events in the HIV-1 life cycle are not intrinsically inefficient in COS-1 cells. These results identify two distinct mechanisms that can restrict the HIV-1 coreceptor activity of US28 in a cell- and coreceptor-dependent manner, and help to explain the existing controversy regarding the ability of US28 to mediate HIV-1 entry.

Apoptosis induced by infection of primary brain cultures with diverse human immunodeficiency virus type 1 isolates: evidence for a role of the envelope.

Apoptosis of neurons and astrocytes is induced by human immunodeficiency type 1 (HIV-1) infection in vitro and has been demonstrated in brain tissue from patients with AIDS. We analyzed a panel of diverse HIV-1 primary isolates for the ability to replicate and induce neuronal and astrocyte apoptosis in primary human brain cultures. Apoptosis was induced three- to eightfold by infection with the blood-derived HIV-1 isolates 89.6, SG3, and ADA. In contrast, the brain-derived HIV-1 isolates YU2, JRFL, DS-br, RC-br, and KJ-br did not induce significant levels of apoptosis. The ability of HIV-1 isolates to induce apoptosis was independent of their replication capacity. Studies of recombinant chimeras between the SG3 and YU2 viruses showed that replacement of the YU2 Env with the SG3 Env was sufficient to confer the ability to induce apoptosis to the YU2 virus. Replacement of the Env V3 regions alone largely conferred the phenotypes of the parental clones. The SG3 Env used CXCR4 and CCR3 as coreceptors for virus entry, whereas YU2 used CCR5 and CCR3. The V3 regions of SG3 and YU2 conferred the ability to use CXCR4 and CCR5, respectively. In contrast, the 3' region of Env, particularly the C3V4 region, was required in conjunction with the V3 region for efficient use of CCR3. These results provide evidence that Env is a major determinant of neurodegenerative mechanisms associated with HIV-1 infection in vitro and raise the possibility that blood-derived viruses which emerge during the late stages of disease may affect disease progression in the central nervous system.

The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly.

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55(gag), the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.

Chemokine receptors in HIV-1 infection of the central nervous system.

Several members of the chemokine receptor are used as coreceptors for HIV-1 infection in the central nervous system (CNS). CCR5 and CCR3 are coreceptors together with CD4 for HIV-1 infection of microglia, the major target for HIV-1 infection in the CNS. Microglia express CXCR4, but their infection by HIV-1 viruses that use only CXCR4 as a coreceptor is relatively inefficient. CXCR4 is also expressed in subpopulations of neurons that are resistant to HIV-1 infection. Additional orphan chemokine receptors that can mediate HIV-1 or SIV entry are expressed in the brain or neurally-derived cell lines, but their role in CNS infection has not been defined. The pattern of chemokine receptor expression in the brain is likely to determine the tropism of HIV-1 for particular CNS target cells and to impact inflammatory and degenerative mechanisms associated with CNS infection.

Ultrastructure of HIV-1 genomic RNA.

The HIV-1 RNA genome is a dimer which consists of two identical strands of RNA linked near their 5' ends by a dimer linkage structure (DLS). We have structurally characterized full-length HIV-1 genomic RNA isolated from HIV-1 virions by electron microscopy. As in other retroviruses, the HIV-1 RNA genome contains a central dimer linkage structure and additional loop structures within each monomer subunit. In contrast to the DLS of other retroviruses, the DLS region of HIV-1 contains a loop of 323 +/- 44 nucleotides. The free 5' ends of the two RNA strands were not visualized, suggesting that the 5' end regions are involved in interstrand complementary base pairing. Computer modeling identified a single stable structure that was consistent with the electron microscopy data. In this model, the two RNA strands are linked at their 5' ends by two contact points derived from "kissing-loop" interactions between r-u5 and SL1 stem-loops and their counterparts on the second strand. These interactions may contribute to the formation of stable HIV-1 RNA dimers in vivo.

Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia.

Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells. T cell line-tropic (T-tropic) HIV-1 viruses use the chemokine receptor CXCR4 as a co-receptor, whereas macrophage-tropic (M-tropic) primary viruses use CCR5 (refs 2-6). Individuals with defective CCR5 alleles exhibit resistance to HIV-1 infection, suggesting that CCR5 has an important role in vivo in HIV-1 replication. A subset of primary viruses can use CCR3 as well as CCR5 as a co-receptor, but the in vivo contribution of CCR3 to HIV-1 infection and pathogenesis is unknown. HIV-1 infects the central nervous system (CNS) and causes the dementia associated with AIDS. Here we report that the major target cells for HIV-1 infection in the CNS, the microglia, express both CCR3 and CCR5. The CCR3 ligand, eotaxin, and an anti-CCR3 antibody inhibited HIV-1 infection of microglia, as did MIP-1beta, which is a CCR5 ligand. Our results suggest that both CCR3 and CCR5 promote efficient infection of the CNS by HIV-1.

The morphology of the immature HIV-1 virion.

Newly released HIV-1 particles exhibit an immature morphology, previously reported to be characterized by a doughnut/ring-shaped structure. In this study we showed that among immature extracellular virus particles not only were particles with doughnut-shaped morphology present, but particles with a crescent morphology were also observed. These particles occurred with different frequencies, depending on whether they were in the cell or in cell-free fractions. The crescent-shaped particles were more abundant in the cell-free fractions, whereas the particles in the cell fraction mainly exhibited doughnut-shaped morphology. The crescent-shaped structure may represent an assembly intermediate.

The role of Gag in human immunodeficiency virus type 1 virion morphogenesis and early steps of the viral life cycle.

The phenotypes of a series of mutant human immunodeficiency virus type 1 proviruses with linker insertion and deletion mutations within the gag coding region were characterized. These mutants, with mutations in the matrix, capsid, and p2 coding regions, produced replication-defective virion particles with defects in the early steps of the viral life cycle. To investigate this phenotype further, the abilities of mutant virion particles to enter T cells, initiate and complete reverse transcription, and transport the newly transcribed proviral DNA were investigated. Only 4 of 10 of the mutants appeared to make wild-type levels of viral DNA. Biochemical analyses of the mutants revealed the middle region of CA as being important in determining virion particle density and sedimentation in velocity gradients. This region also appears to be critical in determining the morphology of mature virion particles by electron microscopy. Particles with aberrant morphology were uninfectious, and only those mutants which displayed cone-shaped cores were capable of carrying out the early steps of the viral life cycle. Thus, the normal morphology of human immunodeficiency virus type 1 appears to be critical to infectivity.

Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo.

The retroviral nucleocapsid (NC) protein is necessary for the specific encapsidation of the viral genomic RNA by the assembling virion. However, it is unclear whether NC contains the determinants for the specific recognition of the viral RNA or instead contributes nonspecific RNA contacts to strengthen a specific contact made elsewhere in the Gag polyprotein. To discriminate between these two possibilities, we have swapped the NC domains of the human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), generating an HIV-1 mutant containing the M-MuLV NC domain and an M-MuLV mutant containing the HIV-1 NC domain. These mutants, as well as several others, were characterized for their abilities to encapsidate HIV-1, M-MuLV, and nonviral RNAs and to preferentially package genomic viral RNAs over spliced viral RNAs. We found that the M-MuLV NC domain mediates the specific packaging of RNAs containing the M-MuLV psi packaging element, while the HIV-1 NC domain confers an ability to package the unspliced HIV-1 RNA over spliced HIV-1 RNAs. In addition, we found that the HIV-1 mutant containing the M-MuLV NC domain exhibited a 20-fold greater ability than wild-type HIV-1 to package a nonviral RNA. These results help confirm the notion that the NC domain specifically recognizes the retroviral genomic RNA during RNA encapsidation.

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.

Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis.

Retroviral capsid (CA) proteins contain a uniquely conserved stretch of 20 amino acids which has been named the major homology region (MHR). To examine the role of this region in human immunodeficiency virus type 1 morphogenesis and replication, four highly conserved positions in the MHR were individually altered by site-directed mutagenesis. Conservative substitution of two invariant residues (glutamine 155 and glutamic acid 159) abolished viral replication and significantly reduced the particle-forming ability of the mutant gag gene products. Conservative substitution of the third invariant residue in the MHR (arginine 167) or of an invariably aromatic residue (tyrosine 164) had only a moderate effect. However, removal of the extended side chains of these amino acids by substitution with alanine prevented viral replication and affected virion morphogenesis. The replacement of tyrosine 164 with alanine substantially impaired viral particle production. By contrast, the substitution of arginine 167 with alanine had only a two- to threefold effect on particle yield but led to the formation of aberrant core structures. The MHR mutant which were severely defective for particle production had a dominant negative effect on particle formation by the wild-type Gag product. The role of the MHR in the incorporation of the Gag-Pol precursor was examined by expressing the Gag and Gag-Pol polyproteins individually from separate plasmids. Only when the two precursor polyproteins were coexpressed did processed Gag and Pol products appear in the external medium. The appearance of these products was unaffected or only moderately affected by substitutions in the MHR of the Gag-Pol precursor, suggesting that the mutant Gag-Pol precursors were efficiently incorporated into viral particles. The results of this study indicate that specific residues within the MHR are required both for human immunodeficiency virus type 1 particle assembly and for the correct assembly of the viral core. However, mutant Gag and Gag-Pol polyproteins with substitutions in the MHR retained the ability to interact with wild-type Gag protein.

Role of vif during packing of the core of HIV-1.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the cell-free infectivity of HIV-1 virus particles. Previous studies have demonstrated that vif increases viral infectivity at the time of virus production, most likely by affecting viral protein processing, virus assembly, or virus maturation. The effect of vif on the assembly and maturation of HIV-1 propagated in CEM, Jurkat, and SupT1 cells was examined by electron microscopy and goniometer analysis. CEM and Jurkat cells are nonpermissive and partially permissive for the replication of vif--defective viruses, respectively, while SupT1 cells are completely permissive. In CEM and Jurkat cultures, the morphology of immature vif+ and vif- virions was similar but immature virus particles were observed at a slightly higher frequency in cultures infected with the vif- virus. At later stages of virus maturation, however, nonhomogeneous packing of the core was detected in the majority of vif- virus particles produced in CEM and Jurkat cells. In the absence of vif, the cone-shaped virus core contained dense material in its broad end but, in contrast to vif+ virions, the material inside its narrow end appeared transparent. The narrow part of the vif- virus core was surrounded by a shell and was attached to the viral envelope by a core-envelope link structure. Vif- virus particles with a lateral body of core material adjacent to the viral envelope were also observed more frequently in CEM and Jurkat cultures. In contrast, in SupT1 cultures the morphology of mature vif+ and vif- virus particles was similar. These results suggest that vif is associated with an effect during the final stages of packing of the viral nucleoprotein core. This effect may be important for the infectivity of HIV-1 virus particles.