Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Adeno-associated virus helper activity of adenovirus DNA binding protein

The requirement for the adenovirus (Ad) single-stranded DNA binding protein (DBP) in the expression of adeno-associated virus (AAV) proteins was studied by specific immunofluorescent staining of infected cells and in vitro translation of RNA from infected cells. The Ad5 mutant ts125, which carries a mutation in the DBP gene, helped AAV as efficiently as the Ad5 wild type (WT) did at both the permissive (32 degrees C) and nonpermissive (40.5 degrees C) temperatures in HeLa and KB cells. Furthermore, at 40.5 degrees C ts125 was as efficient as Ad5WT was in inducing the expression of AAV proteins in a line of Detroit 6 cells which is latently infected with AAV. However, little if any AAV protein was synthesized when coinfections were carried out with Ad5WT in CV-C cells, a monkey cell line that is highly restrictive for human Ad replication unless the cells are also infected with simian virus 40. On the other hand, AAV protein was efficiently produced in CV-C cells in coinfections with the Ad5 mutant hr404, whose growth is unrestricted in CV-C cells and whose mutation also maps in the DBP gene. Finally, preparations of cytoplasmic RNA extracted from CV-C cells infected with AAV and Ad5WT or from CV-C cells infected with AAV, Ad5WT, and simian virus 40 were each capable of directing the in vitro synthesis of abundant amounts of AAV proteins in a rabbit reticulocyte lysate system. These results indicate that the abnormal DBP of ts125 still retains its helper function for AAV replication, but that the molecular feature of the DBP which relates to the monkey cell host range restriction of Ad's may also account for the observed block to AAV protein translation in CV-C cells.     

Herpes Simplex Virus Types 1 and 2 Completely Help Adenovirus-Associated Virus Replication

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

Virus-specific Ribonucleic Acid Synthesis in KB Cells Infected with Herpes Simplex Virus

The production of virus-specific ribonucleic acid (RNA) was investigated in KB cells infected with herpes simplex virus. A fraction of RNA annealable to virus deoxyribonucleic acid (DNA) was found in these cells as early as 3 hr after virus inoculation. Production of this species of RNA increased up to 6 or 7 hr after infection, at which time elaboration of virus messenger RNA (mRNA) declined. At 24 hr after infection, the rate of incorporation of uridine into this RNA was approximately one-half of the rate present at 6 hr after inoculation. Nucleotide analysis of the RNA annealable to virus DNA was compatible with that expected for virus mRNA. Centrifugation showed considerable spread in the size of the virus-induced nucleic acid, the bulk of this RNA sedimenting between 12 and 32S. Incorporation of uridine into cell mRNA, ribosomal precursor RNA, and soluble RNA was suppressed rapidly after infection. As is the case with most other cytocidal viruses investigated to date, virus-induced suppression of cell RNA synthesis appears to be a primary mechanism of cell injury.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.     

Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid

Highly purified, (32)P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of (32)P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonuclease-susceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified (32)P-labeled virus yielded deoxyribonuclease-susceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection.

The capacity of freshly explanted human peripheral blood lymphocytes (PBL) to support the replication of human adenovirus type 2 (Ad2) was investigated. Unlike other types of human cells, PBL were found to be highly nonpermissive. Ad2 adsorbed 30 to 40% of both T and non-T cells. Virus uncoating was very slow and inefficient, resulting in a 40-fold reduction compared with HEp-2 cells. On a population basis, viral DNA synthesis was reduced 460-fold and infectious virus production was reduced 10(6)-fold. Only 0.35% of PBL produced infectious centers, yielding 0.8 PFU per infected cell. Phytohemagglutinin stimulation increased DNA synthesis 23-fold, infectious centers 11-fold, and virus yield 14-fold. We conclude that resting human PBL are highly nonpermissive to Ad2 infection and that phytohemagglutinin can only marginally lift this nonpermissiveness.     

In vivo model of adeno-associated virus vector persistence and rescue.

Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue.